Single cell analysis of transposase accessible chromatin

ABSTRACT

Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/841,411, filed on Apr. 6, 2020, now U.S. Pat. No. 11,155,810 issuedOct. 26, 2021, which is a continuation of U.S. patent application Ser.No. 16/419,555, filed on May 22, 2019, now abandoned, which is acontinuation-in-part of application of U.S. patent application Ser. No.16/206,168, filed on Nov. 30, 2018, now U.S. Pat. No. 11,198,866 issuedDec. 14, 2021, which is a continuation of U.S. patent application Ser.No. 15/842,687, filed on Dec. 14, 2017, now U.S. Pat. No. 10,400,235issued Sep. 3, 2019, which claims priority to U.S. Provisional PatentApplication No. 62/511,905, filed on May 26, 2017, and to U.S.Provisional Patent Application No. 62/527,529, filed on Jun. 30, 2017,each of which applications is entirely incorporated herein by reference.U.S. patent application Ser. No. 16/419,555 is also acontinuation-in-part of PCT Patent Application No. PCT/US2018/034774,filed on May 25, 2018, which claims priority to U.S. patent applicationSer. No. 15/848,714, filed on Dec. 20, 2017, and which claims priorityto U.S. patent application Ser. No. 15/842,550, filed on Dec. 14, 2017,and which claims priority to U.S. patent application Ser. No.15/842,713, filed on Dec. 14, 2017, and which claims priority to U.S.Provisional Patent Application No. 62/650,223, filed on Mar. 29, 2018,each of which applications is entirely incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 26, 2018, isnamed 43487770203SL.txt and is 6,833 bytes in size.

BACKGROUND

Samples may be processed for various purposes, such as identification ofa type of sample of moiety within the sample. The sample may be abiological sample. The biological samples may be processed for variouspurposes, such as detection of a disease (e.g., cancer) oridentification of a particular species. There are various approaches forprocessing samples, such as polymerase chain reaction (PCR) andsequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

Certain applications may benefit from the amplification or sequencing ofspecies obtained from single cells obtained from a much largerpopulation. In some cases, the single cells of interest may be quiterare.

SUMMARY

The present disclosure provides sample preparation techniques that allowsequencing of nucleic acids from single cells of interest. In eukaryoticgenomes, chromosomal DNA winds itself around histone proteins (i.e.,“nucleosomes”), thereby forming a complex known as chromatin. The tightor loose packaging of chromatin contributes to the control of geneexpression. Tightly packed chromatin (“closed chromatin”) is usually notpermissive for gene expression while more loosely packaged, accessibleregions of chromatin (“open chromatin”) is associated with the activetranscription of gene products. Methods for probing genome-wide DNAaccessibility have proven extremely effective in identifying regulatoryelements across a variety of cell types and quantifying changes thatlead to both activation or repression of gene expression.

One such method is the Assay for Transposase Accessible Chromatin withhigh-throughput sequencing (ATAC-seq). The ATAC-seq method probes DNAaccessibility with an artificial transposon, which inserts specificsequences into accessible regions of chromatin. Because the transposasecan only insert sequences into accessible regions of chromatin not boundby transcription factors and/or nucleosomes, sequencing reads can beused to infer regions of increased chromatin accessibility.

Traditional approaches to the ATAC-seq methodology requires large poolsof cells, processes cells in bulk, and result in data representative ofan entire cell population, but lack information about cell-to-cellvariation inherently present in a cell population (see, e.g.,Buenrostro, et al., Curr. Protoc. Mol. Biol., 2015 Jan. 5;21.29.1-21.29.9). While single cell ATAC-seq (scATAC-seq) methods havebeen developed, they suffer from several limitations. For example,scATAC-seq methods that utilize sample pooling, cell indexing, and cellsorting (see, e.g., Cusanovich, et al., Science, 2015 May 22;348(6237):910-14) result in high variability and a low number of readsassociated with any single cell. Other scATAC-seq methods that utilize aprogrammable microfluidic device to isolate single cells and performscATAC-seq in nanoliter reaction chambers (see, e.g., Buenrostro, etal., Nature, 2015 Jul. 23; 523(7561):486-90) are limited by thethroughput of the assay and may not generate personal epigenomicprofiles on a timescale compatible with clinical decision-making.

In an aspect, the present disclosure provides a method for nucleic acidprocessing, comprising: (a) generating a partition comprising: (i) abiological particle comprising chromatin comprising a template nucleicacid molecule; (ii) a plurality of nucleic acid barcode moleculescomprising a common barcode sequence; (iii) a plurality of transposonend nucleic acid molecules comprising a transposon end sequence; and(iv) a plurality of transposase molecules; (b) generating a plurality oftemplate nucleic acid fragments with the aid of a transposase-nucleicacid complex comprising a transposase molecule of the plurality oftransposase molecules and a transposon end oligonucleotide molecule ofthe plurality of transposon end oligonucleotide molecules; and (c)generating a barcoded nucleic acid fragment using a nucleic acid barcodemolecule of the plurality of nucleic acid barcode molecules and atemplate nucleic acid fragment of the plurality of template nucleic acidfragments.

In some embodiments, the barcoded nucleic acid fragment is generated byligation of the nucleic acid barcode molecule with the template nucleicacid fragment, or a derivative thereof.

In some embodiments, the barcoded nucleic acid fragment is generated byamplification of the template nucleic acid fragment, or a derivativethereof, using the nucleic acid barcode molecule as a primer.

In some embodiments, the transposase-nucleic acid complex is generatedin the partition using transposase molecules of the plurality oftransposase molecules and transposon end nucleic acid molecules of theplurality of transposon end nucleic acid molecules.

In some embodiments, the transposase-nucleic acid complex is partitionedinto the partition.

In some embodiments, the plurality of nucleic acid barcode molecules areattached to a bead and wherein the partition further comprises the bead.In some embodiments, the nucleic acid barcode molecules are releasablyattached to the bead. In some embodiments, the method further comprisesreleasing the plurality of nucleic acid barcode molecules from the bead.In some embodiments, the bead is a gel bead. In some embodiments, thegel bead is a degradable gel bead. In some embodiments, the degradablegel bead is degradable upon application of a stimulus. In someembodiments, the stimulus is a chemical stimulus. In some embodiments,the stimulus is a reducing agent. In some embodiments, the partitionfurther comprises the chemical stimulus.

In some embodiments, prior to (b), the template nucleic acid molecule isreleased from the biological particle. In some embodiments, subsequentto (b), the plurality of template nucleic acid fragments is releasedfrom the biological particle.

In some embodiments, the biological particle is a cell. In someembodiments, the biological particle is a nucleus.

In some embodiments, the partition is an aqueous droplet in an emulsion.In some embodiments, the partition is a well.

In some embodiments, the method further comprises releasing or removingthe barcoded nucleic acid fragment or a derivative thereof from thepartition.

In some embodiments, the method further comprises sequencing thebarcoded nucleic acid fragment, or a derivative thereof.

In some embodiments, the plurality of transposon end nucleic acidmolecules further comprise a sequence complementary to a sequence of theplurality of nucleic acid barcode molecules. In some embodiments, thepartition further comprises a plurality of second nucleic acid moleculescomprising a transposon end sequence and a primer sequence, and whereinthe transposase-nucleic acid complex comprises: (i) transposasemolecules of the plurality of transposase molecules; (ii) a transposonend oligonucleotide molecule of the plurality of transposon endoligonucleotide molecules; and (iii) a second nucleic acid molecule ofthe plurality of second nucleic acid molecules. In some embodiments, theplurality of nucleic acid barcode molecules are partiallydouble-stranded and comprise (i) a first strand comprising the commonbarcode sequence and a sequence complementary to a sequence of theplurality of first nucleic acid molecules; and (ii) a second strandcomprising a sequence complementary to the common barcode sequence. Insome embodiments, the plurality of nucleic acid barcode molecules aresingle-stranded and comprise the common barcode sequence and a sequencecomplementary to a sequence in the plurality of transposon end nucleicacid molecules.

In some embodiments, the biological particle further comprises atemplate ribonucleic acid (RNA) molecule, and wherein the method furthercomprises generating a barcoded complementary deoxyribonucleic acid(cDNA) molecule from the template RNA molecule or a derivative thereof.In some embodiments, the barcoded cDNA molecule is generated in thepartition. In some embodiments, the partition comprises a secondplurality of nucleic acid barcode molecules comprising the commonbarcode sequence and a capture sequence. In some embodiments, thecapture sequence is a poly-T sequence. In some embodiments, the capturesequence is a template switching oligo sequence and wherein the barcodedcDNA molecule is generated using a template switching reaction. In someembodiments, the plurality of nucleic acid barcode molecules areattached to a first bead, the second plurality of nucleic acid barcodemolecules are attached to a second bead, and wherein the partitioncomprises the first bead and the second bead. In some embodiments, thefirst bead or the second bead is a magnetic bead. In some embodiments,the first bead is a gel bead and the second bead is a magnetic bead. Insome embodiments, the gel bead comprises the magnetic bead. In someembodiments, the method further comprises degrading the gel bead. Insome embodiments, the first bead is a magnetic bead and the second beadis a gel bead. In some embodiments, the gel bead comprises the magneticbead. In some embodiments, the method further comprises degrading thegel bead. In some embodiments, the plurality of nucleic acid barcodemolecules and the second plurality of nucleic acid barcode molecules areattached to a bead, and wherein the partition further comprises thebead. In some embodiments, the barcoded nucleic acid fragment and thebarcoded cDNA molecule is released from the partition. In someembodiments, the method further comprises sequencing the barcodednucleic acid fragment or a derivative thereof and the barcoded cDNAmolecule or a derivative thereof.

In some embodiments, the method further comprises cleaving one or moremitochondrial nucleic acid fragments, or derivatives thereof, using (i)one or more guide ribonucleic acid molecules (gRNAs) targeted to the oneor more mitochondrial nucleic acid fragments; and (ii) a clusteredregularly interspaced short palindromic (CRISPR) associated (Cas)nuclease. In some embodiments, the partition further comprises the oneor more gRNAs and the Cas nuclease. In some embodiments, the Casnuclease is Cas9.

In another aspect, the present disclosure provides a method ofgenerating barcoded nucleic acid fragments, comprising: (a) providing:(i) a plurality of biological particles, an individual biologicalparticle of the plurality of biological particles comprising chromatincomprising a template nucleic acid; (ii) a plurality of transposon endnucleic acid molecules comprising a transposon end sequence; and (iii) aplurality of transposase molecules; (b) generating a plurality oftemplate nucleic acid fragments in a biological particle of theplurality of biological particles with the aid of a transposase-nucleicacid complex comprising a transposase molecule of the plurality oftransposase molecules and a transposon end nucleic acid molecule of theplurality of transposon end nucleic acid molecules; (c) generating apartition comprising the biological particle comprising the plurality oftemplate nucleic acid fragments and a plurality of nucleic acid barcodemolecules comprising a common barcode sequence; and (d) generating abarcoded nucleic acid fragment using a nucleic acid barcode molecule ofthe plurality of nucleic acid barcode molecules and a template nucleicacid fragment of the plurality of template nucleic acid fragments.

In some embodiments, the barcoded nucleic acid fragment is generated byligation of the nucleic acid barcode molecule with the template nucleicacid fragment, or a derivative thereof.

In some embodiments, the barcoded nucleic acid fragment is generated bynucleic acid amplification of the template nucleic acid fragment, or aderivative thereof, using the nucleic acid barcode molecule as a primer.

In some embodiments, the plurality of nucleic acid barcode molecules areattached to a bead and wherein the partition further comprises the bead.In some embodiments, the nucleic acid barcode molecules are releasablyattached to the bead. In some embodiments, the method further comprisesreleasing the plurality of nucleic acid barcode molecules from the bead.In some embodiments, the bead is a gel bead. In some embodiments, thegel bead is a degradable gel bead. In some embodiments, the degradablegel bead is degradable upon application of a stimulus. In someembodiments, the stimulus is a chemical stimulus. In some embodiments,the stimulus is a reducing agent. In some embodiments, the partitionfurther comprises the chemical stimulus.

In some embodiments, subsequent to (c), the template nucleic acidfragments are released from the biological particle.

In some embodiments, the biological particle is a cell. In someembodiments, the cell is a permeabilized cell.

In some embodiments, the biological particle is a nucleus. In someembodiments, the nucleus is a permeabilized nucleus.

In some embodiments, the partition is an aqueous droplet in an emulsion.In some embodiments, the partition is a well.

In some embodiments, the method further comprises releasing or removingthe barcoded nucleic acid fragment or a derivative thereof from thepartition.

In some embodiments, the method further comprises sequencing thebarcoded nucleic acid fragment, or a derivative thereof.

In some embodiments, the plurality of transposon end nucleic acidmolecules comprise a plurality of first nucleic acid moleculescomprising a transposon end sequence and a sequence complementary to asequence of the plurality of nucleic acid barcode molecules. In someembodiments, the plurality of transposon end nucleic acid moleculesfurther comprise a plurality of second nucleic acid molecules comprisinga transposon end sequence and a primer sequence. In some embodiments,the plurality of nucleic acid barcode molecules are partiallydouble-stranded and comprise a first strand comprising the commonbarcode sequence and a sequence complementary to a sequence of theplurality of first nucleic acid molecules; and a second strandcomprising a sequence complementary to the common barcode sequence. Insome embodiments, the plurality of nucleic acid barcode molecules aresingle-stranded and comprise the common barcode sequence and a sequencecomplementary to a sequence of the plurality of first nucleic acidmolecules.

In some embodiments, the biological particle further comprises atemplate RNA molecule, and wherein the method further comprisesgenerating a barcoded cDNA molecule from the template RNA molecule or aderivative thereof. In some embodiments, the barcoded cDNA molecule isgenerated in the partition. In some embodiments, the partition comprisesa second plurality of nucleic acid barcode molecules comprising thecommon barcode sequence and a capture sequence. In some embodiments, thecapture sequence is a poly-T sequence. In some embodiments, the capturesequence is a template switching oligo sequence and wherein the barcodedcDNA molecule is generated using a template switching reaction. In someembodiments, the plurality of nucleic acid barcode molecules areattached to a first bead, the second plurality of nucleic acid barcodemolecules are attached to a second bead, and wherein the partitioncomprises the first bead and the second bead. In some embodiments, thefirst bead or the second bead is a magnetic bead. In some embodiments,the first bead is a gel bead and the second bead is a magnetic bead. Insome embodiments, the gel bead comprises the magnetic bead. In someembodiments, the method further comprises degrading the gel bead. Insome embodiments, the first bead is a magnetic bead and the second beadis a gel bead. In some embodiments, the gel bead comprises the magneticbead. In some embodiments, the method further comprises degrading thegel bead. In some embodiments, the plurality of nucleic acid barcodemolecules and the second plurality of nucleic acid barcode molecules areattached to a bead, and wherein the partition further comprises thebead. In some embodiments, the barcoded nucleic acid fragment and thebarcoded cDNA molecule is released from the partition. In someembodiments, the method further comprises sequencing the barcodednucleic acid fragment or a derivative thereof and the barcoded cDNAmolecule or a derivative thereof.

In some embodiments, the method further comprises (a) lysing a pluralityof cells in the presence of a cell lysis agent and a blocking agent; and(b) separating a plurality of nuclei from the plurality of lysed cellsto generate the plurality of biological particles, wherein the blockingagent reduces the fraction of sequencing reads corresponding to amitochondrial genome compared to sequencing reads obtained in theabsence of the blocking agent. In some embodiments, the blocking agentis bovine serum albumin (BSA).

In some embodiments, the method further comprises cleaving one or moremitochondrial nucleic acid molecules or derivatives thereof, using (i)one or more guide ribonucleic acid molecules (gRNAs) targeted to the oneor more mitochondrial nucleic acid fragments and (ii) a clusteredregularly interspaced short palindromic (CRISPR) associated (Cas)nuclease. In some embodiments, the Cas nuclease is Cas9. In someembodiments, the mitochondrial nucleic acid molecules are cleaved priorto (c). In some embodiments, the mitochondrial nucleic acid moleculesare cleaved in the partition. In some embodiments, the partitioncomprises the Cas nuclease. In some embodiments, the partition furthercomprises the one or more gRNAs. In some embodiments, the mitochondrialnucleic acid molecules are cleaved subsequent to (d).

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.

FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead.

FIG. 9 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 10 illustrates an exemplary method to produce droplets wherein atleast some of the droplets formed will comprise transposase molecules, asingle cell, and a single gel bead comprising a forked adaptor.

FIGS. 11A-11B illustrate an exemplary method to produce forked adaptorflanked double-stranded template nucleic acid fragments. FIG. 11Aillustrates an exemplary method for the in-partition transposition ofsequencing adaptors into native chromatin while FIG. 11B illustrates anexemplary method for the in-bulk production of a next-generationsequencing compatible library from the fragments produced in FIG. 11A.

FIGS. 12A-12B illustrate exemplary forked adaptors.

FIG. 13 illustrates an exemplary method to produce droplets wherein atleast some of the droplets formed will comprise transposase-nucleic acidcomplexes, a single cell, and a single gel bead comprising a forkedadaptor.

FIGS. 14A-14B illustrate an alternative exemplary method to produceforked adaptor flanked double-stranded template nucleic acid fragments.FIG. 14A illustrates an exemplary method for the in-partition ligationof forked adaptors onto fragments of native chromatin generated by anin-partition transposition reaction. FIG. 14B illustrates an exemplarymethod for the in-bulk production of a next-generation sequencingcompatible library from the fragments produced in FIG. 14A.

FIGS. 15A-15B illustrate additional exemplary forked adaptors andtransposon end sequence containing oligonucleotides.

FIG. 16 illustrates an exemplary method to produce droplets wherein atleast some of the droplets formed will comprise transposase molecules, asingle cell, and a single gel bead comprising a T7-containing adaptor.

FIG. 17 illustrates an exemplary method to produce T7-containing adaptorflanked double-stranded template nucleic acid fragments.

FIG. 18 illustrates an exemplary T7-containing barcoded adaptor.

FIG. 19 illustrates an exemplary method to produce droplets wherein atleast some of the droplets formed will comprise transposase molecules, asingle cell, and a single gel bead comprising a barcoded adaptor.

FIG. 20 illustrates an exemplary scheme for producing barcoded,adapter-flanked nucleic acid fragments.

FIG. 21 illustrates an exemplary partially double-stranded barcodeoligonucleotide releasably attached to a gel bead.

FIG. 22 illustrates a random priming extension reaction scheme.

FIG. 23 illustrates an exemplary method of inserting barcodes into atemplate nucleic acid.

FIGS. 24A-B illustrate an exemplary transposase-nucleic acid complexshowing a transposase, a first partially double-stranded oligonucleotidereleasably attached to a gel bead, the first partially double-strandedoligonucleotide comprising a transposon end sequence, a barcodesequence, and a first primer sequence and a second partiallydouble-stranded oligonucleotide comprising a transposon end sequence anda second primer sequence. In FIG. 24B, the first and the second primersequence are the same.

FIGS. 25A-B illustrates an exemplary barcode oligonucleotides. FIG. 25Aillustrates a partially double-stranded oligonucleotide releasablyattached to a gel bead, the first strand comprising a transposon endsequence, a barcode sequence, and a first primer sequence and a secondstrand comprising a sequence complementary to the transposon endsequence. FIG. 25B illustrates a partially double-strandedoligonucleotide releasably attached to a gel bead, the first strandcomprising a transposon end sequence and a barcode sequence and thesecond strand comprising a sequence complementary to the transposon endsequence

FIG. 26 illustrates an exemplary transposase-nucleic acid complexshowing a transposase, a first double-stranded oligonucleotidecomprising a barcode sequence and a transposon end sequence releasablyattached to a first gel bead and a second double-strandedoligonucleotide comprising a transposon end sequence releasably attachedto a second gel bead.

FIG. 27 illustrates an exemplary method to produce barcoded nucleic acidfragments suitable for next generation sequencing.

FIGS. 28A-B illustrate an exemplary transposase-nucleic acid complex andan exemplary barcoded adaptor releasably attached to a gel bead. FIG.28A illustrates an exemplary transposase-nucleic acid complex showing atransposase, a first double-stranded oligonucleotide comprising atransposon end sequence and a second double-stranded oligonucleotidecomprising a transposon end sequence. FIG. 28B illustrates an exemplarybarcoded adaptor comprising a transposon end sequence, a barcodesequence, and a primer sequence releasably attached to a gel bead.

FIGS. 29A-D illustrate an exemplary transposase-nucleic acid complex andan exemplary barcoded adaptor, which can be releasably attached to a gelbead. FIG. 29A illustrates an exemplary transposase-nucleic acid complexshowing a transposase, a first double-stranded oligonucleotidecomprising a transposon end sequence and a first primer sequence and asecond double-stranded oligonucleotide comprising a transposon endsequence and a second primer sequence. FIG. 28B illustrates an exemplarybarcoded adaptor comprising an adapter sequence, a barcode sequence, anda sequence complementary to the first primer sequence. FIGS. 28C-Dillustrates an exemplary barcoding scheme.

FIGS. 30A-30B illustrate an exemplary barcode oligonucleotide (FIG. 30A)and combination bulk/in-partition barcoding scheme.

FIG. 31 illustrates an exemplary in-partition transposition andbarcoding scheme.

FIGS. 32A-32C illustrate an exemplary barcoding scheme. FIG. 32Aillustrates an exemplary barcode oligonucleotide; FIG. 32B illustratesan exemplary combination of bulk/in-partition barcoding scheme usingCRISPR/Cas-9 mediated cleavage; FIG. 32C illustrates an exemplaryin-partition barcoding scheme using CRISPR/Cas-9 mediated cleavage.

FIGS. 33A-33C illustrate an exemplary barcoding scheme. FIG. 33Aillustrates an exemplary forked barcode oligonucleotide; FIG. 33Billustrates an exemplary combination of bulk/in-partition barcodingscheme using CRISPR/Cas-9 mediated cleavage; FIG. 33C illustrates anexemplary in-partition barcoding scheme using CRISPR/Cas-9 mediatedcleavage.

FIG. 34 illustrates barcoded magnetic beads which can be embedded withina gel bead comprising a barcoded oligonucleotide.

FIG. 35 illustrates exemplary gel bead architectures for single cellATAC-seq (sc-ATAC-seq) plus single cell RNA-seq (scRNA-seq) analyses.BC=barcode.

FIGS. 36A-36B illustrates an exemplary scATAC-seq protocol. FIG. 36Aprovides a schematic of the nuclei preparation of the scATAC-seqprotocol. FIG. 36B illustrates approximate time required for steps inthe scATAC-seq protocol.

FIG. 37 illustrates three exemplary partially-double stranded barcodemolecules suitable for use with a ligation-mediated ATAC-seq scheme asdescribed in Example 9 and 18. In order to assess differences in therate of barcode exchange between the three barcode molecules, twomodifications were developed: a shorter R1 sequence (middle), as well asreplacement of one nucleic acid in the R1 sequence with a uracil(bottom). Shorter R1 (7b); U in R1.

FIGS. 38A-38C illustrate exemplary barnyard plots of mixed human andmouse nuclei demonstrating sequencing reads assembled from scATAC-seqexperiments using the barcode molecules in FIG. 37 . FIG. 38Aillustrates a barnyard plot using original R1 barcode molecules(“control”). FIG. 38B illustrates a barnyard plot using shorter R1barcode molecules (“7b”). FIG. 38C illustrates a barnyard plot using theU-containing barcode molecules (“U40”). These barnyard plots representseparation between the mouse (light grey dots in the upper leftquadrant) and human nucleic acid sequence reads (medium grey dots in thelower right quadrant) from noise (black dots in the lower left quadrant)and doublets (dark grey dots in the upper right quadrant).

FIG. 39 illustrates an exemplary single stranded barcode moleculesuitable for use with a linear amplification-mediated ATAC-seq scheme asdescribed in Example 11. Primers were utilized at either 75 nM, 150 nM,or 250 nM concentrations in the droplet.

FIG. 40 shows a table comparing sequencing metrics generated fromdifferent replicate sample libraries (“A” and “B”) generated usingeither linear amplification or ligation. “A” replicates representreactions done in the same conditions, but using a different user, while“B” replicates represent reactions done in the same conditions by thesame user.

FIGS. 41A-41B illustrate exemplary amounts of detected mouse or humancells and the inferred doublet rate observed from the analysis ofsequencing reads (FIG. 40 ) from a linear amplification ATAC-seq library(using 75 nM, 150 nM, or 250 nM of barcode primer) or ligation-basedATAC-seq library (using the barcode molecules of FIG. 37 ). FIG. 41Aillustrates differences in the number of mouse or human cells detectedusing either a linear amplification or ligation-based ATAC-seq method.FIG. 41B illustrate differences in inferred doublet rate detected usingeither a linear amplification or ligation-based ATAC-seq method.

FIG. 42 illustrates a comparison of the sensitivity of sequencing readsobtained from various ATAC-seq libraries as measured by the medianfragments per cell barcode. The sensitivity of the linear amplificationand ligation methods of library preparation (36 k reads/cell) ascompared to the methods described in Buenrostro, et al., Nature, 2015Jul. 23; 523(7561):48690 using a programmable microfluidics platform(Fluidigm).

FIG. 43 illustrates a library prepared by ligation sequenced toincreased depth (30M reads and 800M reads) compared to FIGS. 40-42demonstrate a significant increase in the sensitivity (as measured bymedian fragments per cell barcode) and reduced noise (as measured byfraction of non-duplicate wasted reads).

FIG. 44 illustrates an exemplary comparison of the total noise(non-duplicate wasted reads (Non-dups”) and mitochondrial-based reads(“Mito”)) in libraries prepared using linear amplification orligation-based ATAC-seq methods.

FIGS. 45A-45B provide an exemplary illustration of the breakdownsequencing reads generated by different library preparation methods.FIG. 45A shows an illustration of the breakdown of reads generated by alinear amplification ATAC-seq library. FIG. 45B shows an illustration ofthe breakdown of reads generated by a ligation-based ATAC-seq library.

FIGS. 46A-46B illustrate an exemplary comparison of read pairs showingthe periodicity of nucleosomes generated from ATAC-seq librariesprepared using either a linear amplification or ligation-based scheme.Nucleosome free fragments are typically observed below 200 bp in length,fragments indicative of a nucleosome periodicity of one areapproximately 200 bp in length, fragments indicative of a nucleosomeperiodicity of two are approximately 400 bp in length, fragmentsindicative of a nucleosome periodicity of two are 600 bp in length, andso forth. FIG. 46A illustrates insert lengths of fragments from alibrary prepared by linear amplification. FIG. 46B illustrates insertlengths of fragments from a library prepared by ligation.

FIGS. 47A-47B illustrate an exemplary enrichment of transcription startsites (TSS) or analysis of CTCF (CCCTC-binding factor) sites observed inATAC-seq libraries prepared by linear amplification or ligation-basedmethodologies vs a traditional bulk ATAC-seq library preparation using50,000 nuclei. FIG. 47A illustrates exemplary enrichment of TSS data.FIG. 47B illustrates enrichment of CTCF sites.

FIG. 48 shows a table comparing exemplary sequencing metrics obtainedfrom linear amplification-based ATAC-seq libraries prepared usingdifferent polymerases: a Phusion® DNA polymerase, a KAPA HiFi DNApolymerase (in combination with betaine), a Deep Vent DNA polymerase, aswell as a library prepared by ligation.

FIG. 49 shows a table providing exemplary parameters of 1,000 pLdroplets vs. smaller—400 pL droplets. The benefits of using a smallerdroplet size include—2-fold decrease in Poisson derived cell doubletrate and an—2-fold increase in cell throughput.

FIG. 50 illustrates protocols for ATAC-seq as described herein comparedto data from (1) typical high quality traditional bulk ATAC-seqprotocols; (2) Cusanovich, et al., Science, 2015 May 22;348(6237):910-14; (3) Buenrostro, et al., Nature, 2015 Jul. 23;523(7561):486-90; (4) ideal sequencing metrics from an ATAC-seqexperiment; (5) data obtained using the methods described herein(“10×”).

FIGS. 51A-51B shows the results of scRNA-seq and scATAC-seq analysis ofnucleic acids in a peripheral blood mononuclear cell (PBMC) sample. FIG.51A shows an exemplary scatterplot produced using t-DistributedStochastic Neighbor Embedding (tSNE), allowing visualization of RNAtranscripts of different subpopulations cell types in a peripheral bloodmononuclear cell (PBMC) sample. FIG. 51B shows an exemplary scatterplotproduced using t-Distributed Stochastic Neighbor Embedding (tSNE),allowing visualization of ATAC-seq data of different subpopulations celltypes in a peripheral blood mononuclear cell (PBMC) sample.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent of a cell. The biologicalparticle may be or may include DNA, RNA, organelles, proteins, or anycombination thereof. The biological particle may be or include achromosome or other portion of a genome. The biological particle may beor may include a matrix (e.g., a gel or polymer matrix) comprising acell or one or more constituents from a cell (e.g., cell bead), such asDNA, RNA, organelles, proteins, or any combination thereof, from thecell. The biological particle may be obtained from a tissue of asubject. The biological particle may be a hardened cell. Such hardenedcell may or may not include a cell wall or cell membrane. The biologicalparticle may include one or more constituents of a cell, but may notinclude other constituents of the cell. An example of such constituentsis a nucleus or an organelle. A cell may be a live cell. The live cellmay be capable of being cultured, for example, being cultured whenenclosed in a gel or polymer matrix, or cultured when comprising a gelor polymer matrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA. The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

Single Cell Assay for Transposase Accessible Chromatin using Sequencing(ATAC-seq)

Disclosed herein, in some embodiments, are methods for nucleic acidprocessing. A method for nucleic acid processing may comprise generatinga partition (e.g., a droplet or well) comprising: (i) a biologicalparticle (e.g., a cell or nucleus) comprising chromatin comprising atemplate nucleic acid molecule; (ii) a plurality of nucleic acidmolecules comprising a common barcode sequence; (iii) a plurality oftransposon end nucleic acid molecules comprising a transposon endsequence; and (iv) a plurality of transposase molecules. A plurality oftemplate nucleic acid fragments may then be generated with the aid of atransposase-nucleic acid complex comprising a transposase molecule ofthe plurality of transposase molecules and a transposon endoligonucleotide molecule of the plurality of transposon endoligonucleotide molecules. A barcoded nucleic acid fragment may then begenerated using a nucleic acid barcode molecule of the plurality ofnucleic acid barcode molecules and a template nucleic acid fragment ofthe plurality of template nucleic acid fragments.

The present disclosure also discloses a method of generating barcodednucleic acid fragments, comprising providing: (i) a plurality ofbiological particles (e.g., cells or nuclei), an individual biologicalparticle of the plurality of biological particles comprising chromatincomprising a template nucleic acid; (ii) a plurality of transposon endnucleic acid molecules comprising a transposon end sequence; and (iii) aplurality of transposase molecules. A plurality of template nucleic acidfragments may then be generated in a biological particle of theplurality of biological particles with the aid of a transposase-nucleicacid complex comprising a transposase molecule of the plurality oftransposase molecules and a transposon end nucleic acid molecule of theplurality of transposon end nucleic acid molecules. A partition may thenbe generated, where the partition comprises the biological particlecomprising the plurality of template nucleic acid fragments and aplurality of nucleic acid barcode molecules comprising a common barcodesequence. A barcoded nucleic acid fragment may then be generated using anucleic acid barcode molecule of the plurality of nucleic acid barcodemolecules and a template nucleic acid fragment of the plurality oftemplate nucleic acid fragments.

In some embodiments, the transposase is a Tn5 transposase. In someembodiments, the transposase is a mutated, hyperactive Tn5 transposase.In some embodiments, the transposase is a Mu transposase. In someembodiments, the partitions disclosed in (a) further comprise cell lysisreagents; and/or (b) reagent and buffers necessary to carry out one ormore reactions.

In some embodiments, a partition (e.g., a droplet or well) comprises asingle cell and is processed according to the methods described herein.In some embodiments, a partition comprises a single cell and/or a singlenucleus. The single cell and/or the single nucleus may be partitionedand processed according to the methods described herein. In some cases,the single nucleus may be a component of a cell. In some embodiments, apartition comprises chromatin from a single cell or single nucleus(e.g., a single chromosome or other portion of the genome) and ispartitioned and processed according to the methods described herein. Insome embodiments, the transposition reactions and methods describedherein are performed in bulk and biological particles (e.g.,nuclei/cells/chromatin from single cells) are then partitioned such thata plurality of partitions is singly occupied by a biological particle(e.g., a cell, cell nucleus, chromatin, or cell bead). For example, aplurality of biological particles may be partitioned into a plurality ofpartitions such that partitions of the plurality of partitions comprisea single biological particle.

In some embodiments, the oligonucleotides described herein comprise atransposon end sequence. In some embodiments, the transposon endsequence is a Tn5 or modified Tn5 transposon end sequence. In someembodiments, the transposon end sequence is a Mu transposon endsequence. In some embodiments, the transposon end sequence has asequence of: AGATGTGTATAAGAGACA (SEQ ID NO: 1).

In some embodiments, the oligonucleotides described herein comprise anRI sequencing priming region. In some embodiments, the R1 sequencingprimer region has a sequence of TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT(SEQ ID NO: 2), or some portion thereof. In some embodiments, the R1sequencing primer region has a sequence ofTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (SEQ ID NO: 3), or some portionthereof. In some embodiments, the oligonucleotides described hereincomprise a partial R1 sequence. In some embodiments, the partial R1sequence is ACTACACGACGCTCTTCCGATCT (SEQ ID NO: 4). In some embodiments,the oligonucleotides described herein comprise an R2 sequencing primingregion. In some embodiments, the R2 sequencing primer region has asequence of GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 5), or someportion thereof. In some embodiments, the R2 sequencing primer regionhas a sequence of GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO: 6), orsome portion thereof. In some embodiments, the oligonucleotidesdescribed herein comprise a T7 promoter sequence. In some embodiments,the T7 promoter sequence is TAATACGACTCACTATAG (SEQ ID NO: 7). In someembodiments, the oligonucleotides described herein comprise a region atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-7. In someembodiments, the oligonucleotides described herein comprise a P5sequence. In some embodiments, the oligonucleotides described hereincomprise a P7 sequence. In some embodiments, the oligonucleotidesdescribed herein comprise a sample index sequence.

In some embodiments, the oligonucleotides described herein are attachedto a solid support (e.g., a solid or semi-solid particle such as abead). In some embodiments, the oligonucleotides described herein areattached to a bead. In some embodiments, the bead is a gel bead. In someembodiments, the oligonucleotides described herein are releasablyattached to a bead (e.g., a gel bead). In some embodiments, theoligonucleotides described herein are single-stranded and the firststrand attached to a gel bead. In some embodiments, the oligonucleotidesdescribed herein are double-stranded or partially double-strandedmolecules and the first strand is releasably attached to a bead. In someembodiments, the oligonucleotides described herein are double-strandedor partially double-stranded molecules and the second strand isreleasably attached to a bead. In some embodiments, the oligonucleotidesdescribed herein are double-stranded or partially double-strandedmolecules and both the first and the second strand are releasablyattached to a bead or a collection of beads.

In some embodiments, the solid support (e.g., bead, such as a gel bead)comprises a plurality of first oligonucleotides and a plurality ofsecond oligonucleotides. In some embodiments, the firstoligonucleotides, the second oligonucleotides, or the combinationthereof are releasably attached to a bead. In some embodiments, thefirst oligonucleotides, the second oligonucleotides, or the combinationthereof are double-stranded or partially double-stranded molecules andthe first strand is releasably attached to a bead. In some embodiments,the first oligonucleotides, the second oligonucleotides, or thecombination thereof are double-stranded or partially double-strandedmolecules and the second strand is releasably attached to abead. In someembodiments, the first oligonucleotides, the second oligonucleotides, orthe combination thereof are double-stranded or partially double-strandedmolecules and the first strand and the second strand are releasablyattached to abead. In some embodiments, the oligonucleotides, the firstoligonucleotides, the second oligonucleotides, or a combination there ofare bound to a magnetic particle. In some embodiments, the magneticparticle is embedded in a solid support (e.g., a bead, such as a gelbead).

In some embodiments, the first oligonucleotides are capable of coupling(e.g., by nucleic acid hybridization) to DNA molecules and the secondoligonucleotides are capable of coupling (e.g., by nucleic acidhybridization) to RNA molecules (e.g., mRNA molecules). Examples ofoligonucleotide architecture are provided in FIG. 35 . In someembodiments, the first oligonucleotide comprises a P5 adaptor sequence,a barcode sequence, and an RI sequence or partial RI primer sequence. Insome embodiments, the second oligonucleotide comprises a RI sequence orpartial RI primer sequence, a barcode sequence, a unique molecularidentifier (LIMI) sequence, and a poly(dT) sequence. In someembodiments, the second oligonucleotide comprises a RI sequence orpartial RI primer sequence, a barcode sequence, a unique molecularidentifier (UNIT) sequence, and a switch oligo.

In some embodiments, the first oligonucleotide comprises a P5 adaptorsequence, a barcode sequence, and an R1 sequence or partial R1 primersequence and is partially double-stranded, and the secondoligonucleotide comprises a R1 or partial R1 primer sequence, a barcodesequence, a unique molecular identifier (UNIT) sequence, and a poly(dT)sequence. In some embodiments, the first oligonucleotide comprises a P5adaptor sequence, a barcode sequence, and an R1 or partial R1 primersequence and is single-stranded, and the second oligonucleotidecomprises a R1 or partial R1 sequence, a barcode sequence, a uniquemolecular identifier (UMI) sequence, and a template switching oligosequence.

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, and/or partitioning of one or moreparticles (e.g., biological particles, macromolecular constituents ofbiological particles, beads, reagents, etc.) into discrete compartmentsor partitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be, for example, a wellor a droplet in an emulsion. A partition may comprise one or more otherpartitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles and/ormacromolecular constituents thereof. For example, a partition maycomprise one or more cells, nuclei, chromatins, and/or cell beads. Forinstance, a partition may comprise one or more cell beads. A cell beadcan be a biological particle and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the biological particle andprecursors capable of being polymerized or gelled. A partition maycomprise one or more solid or semi-solid particles. For example, apartition may comprise one or more beads, such as one or more gel beads.In some cases, a partition may include a single gel bead, a single cellbead, or both a single cell bead and single gel bead. A partition mayinclude one or more reagents (e.g., as described herein). Uniqueidentifiers, such as barcodes, may be injected into the dropletsprevious to, subsequent to, or concurrently with droplet generation,such as via a microcapsule (e.g., bead), as described elsewhere herein.The barcodes may comprise nucleic acid sequences. Such nucleic acidbarcode sequences may be components of nucleic acid barcode molecules,which may be coupled (e.g., releasably coupled) to a solid or semi-solidparticle such as a bead. In some cases, a partition may be unoccupied.For example, a partition may not comprise a bead or a biologicalparticle. Microfluidic devices comprising one or more channels (e.g.,microfluidic channel networks of a chip) can be utilized to generatepartitions as described herein. For example, a first fluid and a secondfluid that is immiscible with the first fluid may be flowed to a dropletgeneration junction, and a droplet of the first fluid may be generatedwithin the second fluid. Alternative mechanisms may also be employed inthe partitioning of individual biological particles, including porousmembranes through which aqueous mixtures of cells are extruded intonon-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of biological particles per partition, number of beads perpartition, etc.). For example, partition occupancy can be controlled byproviding the aqueous stream at a certain concentration and/or flow rateof particles. To generate single biological particle partitions, therelative flow rates of the immiscible fluids can be selected such that,on average, the partitions may contain less than one biological particleper partition in order to ensure that those partitions that are occupiedare primarily singly occupied. In some cases, partitions among aplurality of partitions may contain at most one biological particle(e.g., cell bead, chromatin, DNA, cell, cell nucleus, or other cellularmaterial). In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) maybe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles, (e.g., as describedherein). The channel structure 100 can include channel segments 102,104, 106 and 108 communicating at a channel junction 110. In operation,a first aqueous fluid 112 that includes suspended biological particles(or cells) 114 may be transported along channel segment 102 intojunction 110, while a second fluid 116 that is immiscible with theaqueous fluid 112 is delivered to the junction 110 from each of channelsegments 104 and 106 to create discrete droplets 118, 120 of the firstaqueous fluid 112 flowing into channel segment 108, and flowing awayfrom junction 110. The channel segment 108 may be fluidically coupled toan outlet reservoir where the discrete droplets can be stored and/orharvested. A discrete droplet generated may include an individualbiological particle 114 (such as droplets 118). A discrete dropletgenerated may include more than one individual biological particle 114(not shown in FIG. 1 ). A discrete droplet may contain no biologicalparticle 114 (such as droplet 120). Each discrete partition may maintainseparation of its own contents (e.g., individual biological particle114) from the contents of other partitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles,cell beads, and/or solid or semi-solid particles such as gel beads) thatmeet at a channel junction. Fluid may be directed to flow along one ormore channels or reservoirs via one or more fluid flow units. A fluidflow unit can comprise compressors (e.g., providing positive pressure),pumps (e.g., providing negative pressure), actuators, and the like tocontrol flow of the fluid. Fluid may also or otherwise be controlled viaapplied pressure differentials, centrifugal force, electrokineticpumping, vacuum, capillary or gravity flow, or the like.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of singly-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein may result inpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2 ). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules anda biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles (e.g., as described herein) may beencapsulated within a microcapsule that comprises an outer shell, layeror porous matrix in which is entrained one or more individual biologicalparticles or small groups of biological particles. The microcapsule mayinclude other reagents. Encapsulation of biological particles may beperformed by a variety of processes. Such processes may combine anaqueous fluid containing the biological particles with a polymericprecursor material that may be capable of being formed into a gel orother solid or semi-solid matrix upon application of a particularstimulus to the polymer precursor. Such stimuli can include, forexample, thermal stimuli (e.g., either heating or cooling),photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g.,through crosslinking, polymerization initiation of the precursor (e.g.,through added initiators)), mechanical stimuli, or a combinationthereof.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1 , may be readily used in encapsulating cells asdescribed herein. In particular, and with reference to FIG. 1 , theaqueous fluid 112 comprising (i) the biological particles 114 and (ii)the polymer precursor material (not shown) is flowed into channeljunction 110, where it is partitioned into droplets 118, 120 through theflow of non-aqueous fluid 116. In the case of encapsulation methods,non-aqueous fluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the biological particles (e.g., cells) 114.Although described in terms of polyacrylamide encapsulation, otheractivatable' encapsulation compositions may also be employed in thecontext of the methods and compositions described herein. For example,formation of alginate droplets followed by exposure to divalent metalions (e.g., Ca²⁺ ions), can be used as an encapsulation process usingthe described processes. Likewise, agarose droplets may also betransformed into capsules through temperature based gelling (e.g., uponcooling, etc.).

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as dithiothreitol (DTT) or the like, to cleave disulfidebonds that cross-link the polymer matrix. See, for example, U.S. PatentApplication Publication No. 2014/0378345, which is entirely incorporatedherein by reference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around and/orwithin the biological particle. The polymer or gel may be diffusivelypermeable to chemical or biochemical reagents. The polymer or gel may bediffusively impermeable to macromolecular constituents of the biologicalparticle. In this manner, the polymer or gel may act to allow thebiological particle to be subjected to chemical or biochemicaloperations while spatially confining the macromolecular constituents toa region of the droplet defined by the polymer or gel. The polymer orgel may include one or more of disulfide cross-linked polyacrylamide,agarose, alginate, polyvinyl alcohol, polyethylene glycol(PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, otheracrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, orelastin. The polymer or gel may comprise any other polymer or gel. Acell comprising a polymer or gel matrix may be referred to as a “cellbead.”

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. For example, polymerization or gelling may be brought aboutby a change in salinity, pH, temperature, or pressure. The polymer orgel may be stable in alkaline conditions or at elevated temperature. Thepolymer or gel may have mechanical properties similar to the mechanicalproperties of the bead. For instance, a polymer or gel matrix may be ofa similar size to the bead. The polymer or gel may have a mechanicalstrength (e.g. tensile strength) similar to that of the bead. Thepolymer or gel may be of a lower density than an oil. The polymer or gelmay be of a density that is roughly similar to that of a buffer. Thepolymer or gel may have a tunable pore size. The pore size may be chosento, for instance, retain denatured nucleic acids. The pore size may bechosen to maintain diffusive permeability to exogenous chemicals such assodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.The polymer or gel may be biocompatible. The polymer or gel may maintainor enhance cell viability. The polymer or gel may be biochemicallycompatible. The polymer or gel may be polymerized and/or depolymerizedthermally, chemically, enzymatically, and/or optically.

The polymer or gel may comprise poly(acrylamide-co-acrylic acid)crosslinked with disulfide linkages. The preparation of the polymer orgel may comprise a two-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such as one ormore barcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example, as components of a nucleic acid molecule(e.g., an oligonucleotide). Barcodes may be delivered to a partition viaany suitable mechanism. For example, barcoded nucleic acid molecules canbe delivered to a partition such as a droplet or well via a microcapsuleor bead. A microcapsule, in some instances, can comprise a bead. Beadsare described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule or bead and then released from themicrocapsule or bead. For example, a bead may comprise a plurality ofnucleic acid barcode molecules releasably coupled thereto. Release ofthe barcoded nucleic acid molecules can be passive (e.g., by diffusionout of the microcapsule or bead). In addition or alternatively, releasefrom the microcapsule or bead can be upon application of a stimuluswhich allows the barcoded nucleic acid nucleic acid molecules todissociate or to be released from the microcapsule or bead. Suchstimulus may disrupt the microcapsule or bead, an interaction thatcouples the barcoded nucleic acid molecules to or within themicrocapsule or bead, or both. Such stimulus can include, for example, athermal stimulus, photo-stimulus, chemical stimulus (e.g., change in pHor use of a reducing agent(s)), a mechanical stimulus, a radiationstimulus; a biological stimulus (e.g., enzyme), or any combinationthereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., beads comprising nucleic acid barcode molecules,oligonucleotides, or molecular tags) along the channel segment 201 intojunction 210. The plurality of beads 214 may be sourced from asuspension of beads. For example, the channel segment 201 may beconnected to a reservoir comprising an aqueous suspension of beads 214.The channel segment 202 may transport the aqueous fluid 212 thatincludes a plurality of biological particles 216 along the channelsegment 202 into junction 210. The plurality of biological particles 216may be sourced from a suspension of biological particles. For example,the channel segment 202 may be connected to a reservoir comprising anaqueous suspension of biological particles 216. In some instances, theaqueous fluid 212 in either the first channel segment 201 or the secondchannel segment 202, or in both segments, can include one or morereagents, as further described below. A second fluid 218 that isimmiscible with the aqueous fluid 212 (e.g., oil) can be delivered tothe junction 210 from each of channel segments 204 and 206. Upon meetingof the aqueous fluid 212 from each of channel segments 201 and 202 andthe second fluid 218 from each of channel segments 204 and 206 at thechannel junction 210, the aqueous fluid 212 can be partitioned asdiscrete droplets 220 in the second fluid 218 and flow away from thejunction 210 along channel segment 208. The channel segment 208 maydeliver the discrete droplets to an outlet reservoir fluidly coupled tothe channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets (e.g., droplets comprisingbeads and/or biological particles) may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to be formed such that itincludes a single bead and a single biological particle. Such regularflow profiles may permit the droplets to have an occupancy (e.g.,droplets having beads and biological particles) greater than 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flowprofiles and devices that may be used to provide such regular flowprofiles are provided in, for example, U.S. Patent Publication No.2015/0292988, which is entirely incorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a bead 214 comprising a barcode or other reagent. For example,bead 214 may comprise a plurality of nucleic acid barcode molecules eachcomprising a common barcode sequence. A discrete droplet generated mayinclude both an individual biological particle and a barcode carryingbead, such as droplets 220. In some instances, a discrete droplet mayinclude more than one individual biological particle or no biologicalparticle. In some instances, a discrete droplet may include more thanone bead or no bead. A discrete droplet may be unoccupied (e.g., nobeads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less. A bead may comprise natural and/or syntheticmaterials. For example, a bead can comprise a natural polymer, asynthetic polymer or both natural and synthetic polymers. Examples ofnatural polymers include proteins and sugars such as deoxyribonucleicacid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins,enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan,dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin,shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gumkaraya, agarose, alginic acid, alginate, or natural polymers thereof.Examples of synthetic polymers include acrylics, nylons, silicones,spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene,polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations(e.g., co-polymers) thereof. Beads may also be formed from materialsother than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules (e.g., nucleic acid barcode molecules) coupled toa given bead and/or sequences that are different across all nucleic acidmolecules coupled to the given bead. The nucleic acid molecule may beincorporated into the bead and/or may be attached to a surface of thebead.

In some cases, the nucleic acid molecule (e.g., nucleic acid barcodemolecule) can comprise a functional sequence, for example, forattachment to a sequencing flow cell, such as, for example, a P5sequence for Illumina® sequencing. In some cases, the nucleic acidmolecule (e.g., nucleic acid barcode molecule) or derivative thereof(e.g., oligonucleotide or polynucleotide generated from the nucleic acidmolecule) can comprise another functional sequence, such as, forexample, a P7 sequence for attachment to a sequencing flow cell forIllumina sequencing. In some cases, the nucleic acid molecule cancomprise a barcode sequence. In some cases, the primer can furthercomprise a unique molecular identifier (UMI). In some cases, the primercan comprise an R1 primer sequence for Illumina sequencing. In somecases, the primer can comprise an R2 primer sequence for Illuminasequencing. Examples of such nucleic acid molecules (e.g.,oligonucleotides, polynucleotides, etc.) and uses thereof, as may beused with compositions, devices, methods and systems of the presentdisclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and2015/0376609, each of which is entirely incorporated herein byreference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820 can be released from the bead 804in the partition. By way of example, in the context of analyzing sampleRNA, the poly-T segment (e.g., 812) of one of the released nucleic acidmolecules (e.g., 802) can hybridize to the poly-A tail of a mRNAmolecule. Reverse transcription may result in a cDNA transcript of themRNA, but which transcript includes each of the sequence segments 808,810, 816 of the nucleic acid molecule 802. Because the nucleic acidmolecule 802 comprises an anchoring sequence 814, it will more likelyhybridize to and prime reverse transcription at the sequence end of thepoly-A tail of the mRNA. Within any given partition, all of the cDNAtranscripts of the individual mRNA molecules may include a commonbarcode sequence segment 810. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 812 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the biological particle (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include 13-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),13-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes. FIG. 49 shows exemplary parametersof 1,000 pL droplets vs. smaller—400 pL droplets. In some cases, the useof a smaller droplet size may result in a decrease in Poisson derivedcell doublet rate. The use of a smaller droplet size may also result inan ˜2-fold increase in cell throughput.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles's contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with thebiological particles described above, other reagents can also beco-partitioned with the biological particles, including, for example,DNase and RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated biological particles, thebiological particles may be exposed to an appropriate stimulus torelease the biological particles or their contents from a co-partitionedmicrocapsule. For example, in some cases, a chemical stimulus may beco-partitioned along with an encapsulated biological particle to allowfor the degradation of the microcapsule and release of the cell or itscontents into the larger partition. In some cases, this stimulus may bethe same as the stimulus described elsewhere herein for release ofnucleic acid molecules (e.g., oligonucleotides) from their respectivemicrocapsule (e.g., bead). In alternative aspects, this may be adifferent and non-overlapping stimulus, in order to allow anencapsulated biological particle to be released into a partition at adifferent time from the release of nucleic acid molecules into the samepartition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNAse, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxylnosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particles, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2 ). Insome aspects, the unique identifiers are provided in the form of nucleicacid molecules (e.g., oligonucleotides) that comprise nucleic acidbarcode sequences that may be attached to or otherwise associated withthe nucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4 ). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2 ). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, a. The expansion angle, a, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, Rd, may be predicted bythe following equation for the aforementioned geometric parameters ofh_(o), w, and α:R _(d)≈0.44(1+2.2√{square root over (tan α)}w/h ₀)h ₀/√{square root over(tan α)}

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 μm, h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, a, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and a, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, a (not shown in FIG. 6 )at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and a, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6 ) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., zlh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4 ), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7 ). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂ may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, (3, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Methods and Systems for Reducing or Removing Mitochondrial DNA

Cells comprise both nuclear DNA (e.g., DNA comprising chromatin) andmitochondrial DNA. In the tagmentation methods described herein, bothnuclear DNA and mitochondrial DNA may be susceptible to tagmentation.Depending on the cell type, a method for measuring accessible chromatinmay yield a plurality of sequencing reads, where between about 20 and80% of the sequencing reads may be attributable to mitochondrial DNA.Such reads may be discarded if nuclear DNA (e.g., accessible chromatin)is of interest. Techniques to either eliminate or reduce mitochondrialDNA prior to tagmentation or to remove or reduce tagmented mitochondrialDNA fragments prior to sequencing may result in enhancement inchromatin-attributable data.

The present disclosure further provides a method of enhancing nuclearDNA within a sample. The method may comprise reducing or removingmitochondrial DNA. In some cases, the method may comprise providing asample comprising both mitochondrial DNA and nuclear DNA, and removingmitochondrial DNA from or reducing the relative amount of mitochondrialDNA in the sample prior to analyzing cells or nuclei within the samplefor accessible chromatin (e.g., tagmentation). In some cases, the methodmay comprise providing a sample comprising both mitochondrial DNA andnuclear DNA; subjecting the sample to analysis for accessible chromatin(e.g., as described herein), thereby generating a processed samplecomprising both tagmented mitochondrial DNA fragments and tagmentednuclear DNA fragments; and removing tagmented mitochondrial DNAfragments from the processed sample or reducing the relative amount oftagmented mitochondrial DNA fragments within the processed sample.

In some cases, whole cells may be used as the input material for anATAC-seq analysis (e.g., as described herein). In some cases, nuclei maybe used as the input material for an ATAC-seq analysis. For example,nuclei may be used as the input material where mitochondrial DNA isremoved prior to tagmentation of genomic DNA. In this approach, wholecells may be gently lysed to remove their cytoplasmic membrane whilstleaving the nuclear membrane and nucleoprotein packaging of the genomeintact. In some cases, a lysis buffer used to lyse whole cells maycomprise one or more of Tris-HCl, NaCl, magnesium ions (e.g., MgCl₂),and a lysis agent. A lysis agent may be, for example, NP40, Tween-20,Digitonin, Nonident P40, or DBDM. Mitochondria may be washed away alongwith other cellular debris during subsequent centrifugation steps toyield a suspension of nuclei.

In some cases, mitochondrial membranes may be lysed along with theircytoplasmic counterparts. This may result in mitochondrial DNAnon-specifically binding to nuclear membranes during this step and thusbeing carried into a subsequent tagmentation reaction. A blocking agent(e.g., a blocking protein) may be used to prevent nonspecific binding ofmitochondrial DNA to nuclei membranes during cell lysis. A blockingagent may not inhibit downstream biochemistry. In some cases, a blockingagent may be selected from the group consisting of, for example, asingle purified protein (e.g., bovine serum albumin or casein), a singlepurified glycoprotein, a milk protein, fish gelatin, a normal serum(e.g., fetal calf serum, rabbit serum, goat serum, or the like), abuffer such as ThermoFisher Scientific's SuperBlock, andpolyvinylpyrrolidone (PVP). For example, the blocking agent (e.g.,blocking protein) may be bovine serum albumin (BSA), an inexpensive andreadily available isolated protein. The blocking protein (e.g., BSA) maybe added to a detergent formulation used during cell lysis. The blockingprotein (e.g., BSA) may block available binding sites on nucleimembrane, thereby preventing nonspecific binding of mitochondrial DNA.The blocking agent (e.g., blocking protein) may be provided in anyuseful concentration. The blocking agent may be provided in a solutioncomprising a buffer. After providing a blocking agent to a sample (e.g.,a solution, partition, or plurality of partitions, such as a pluralityof aqueous droplets) comprising one or more cells and/or nuclei, thesample may be agitated and/or centrifuged one or more times, and/orundergo one or more washing steps. During subsequent processing (e.g.,centrifugation), cell debris and mitochondrial DNA may be washed away(e.g., in a supernatant). This approach may reduce the amount ofsequencing reads associated with mitochondrial DNA in an ATAC-seqlibrary by at least about 5%. For example, the amount of sequencingreads associated with mitochondrial DNA may be reduced by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, or by 100%. In an example, the amount ofsequencing reads associated with mitochondrial DNA may be reduced by atleast about 50%, such as from about 30% to about 60%. In some cases, thefraction of sequencing reads associated with mitochondrial DNA of aplurality of sequencing reads derived from a sample (e.g., a solution,partition, or plurality of partitions comprising one or more cellsand/or nuclei) may be reduced to be less than about 60%. For example,the fraction of sequencing reads associated with mitochondrial DNA maybe reduced to less than about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%, or less of the total sequencing reads derivedfrom a sample. In an example, the fraction of sequencing readsassociated with mitochondrial DNA may be reduced to between about 1-5%of the total sequencing reads derived from a sample.

In some cases, a method of generating barcoded nucleic acid fragmentscomprises: (a) lysing a plurality of cells in the presence of a celllysis agent and a blocking agent to generate a plurality of lysed cells;(b) separating a plurality of nuclei from the plurality of lysed cellsto generate a plurality of biological particles, where an individualbiological particle (e.g., cell) comprises a non-template nucleic acidand chromatin comprising a template nucleic acid; (c) providing (i) aplurality of transposon end nucleic acid molecules comprising atransposon end sequence and (ii) a plurality of transposase nucleic acidmolecules; (d) generating a plurality of template nucleic acid fragmentsin a biological particle of the plurality of biological particles withthe aid of a transposase-nucleic acid complex comprising a transposasenucleic acid molecule of the plurality of transposase nucleic acidmolecules and a transposon end nucleic acid molecule of the plurality oftransposon end nucleic acid molecules; (e) generating a partition (e.g.,a droplet or well) comprising the biological particle comprising theplurality of template nucleic acid fragments and a plurality of barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules)comprising a barcode sequence; and (1) generating a barcoded templatenucleic acid fragment using a barcode oligonucleotide molecule (e.g.,nucleic acid barcode molecule) of the plurality of barcodeoligonucleotide molecule and a template nucleic acid fragment of theplurality of template nucleic acid fragments. In some cases, theblocking agent may comprise bovine serum albumin (BSA). For example, theblocking agent may comprise a solution of 3% BSA in phosphate bufferedsaline. Lysing the plurality of cells may comprise providing a lysisbuffer comprising the cell lysis agent to the plurality of cells. Insome cases, the biological particles may be nuclei, which may beisolated from lysed cells by, e.g., washing the lysed cells. In somecases, the method further comprises generating sequencing reads.Application of the method may reduce the fraction of sequencing readsderiving from mitochondrial DNA relative to the total number ofsequencing reads generated. In some cases, the template nucleic acid maycomprise chromatin.

In some cases, a method of generating barcoded nucleic acid fragmentscomprises: (a) lysing a plurality of cells in the presence of a celllysis agent and a blocking agent to generate a plurality of lysed cells;(b) separating a plurality of nuclei from the plurality of lysed cellsto generate a plurality of biological particles, where an individualbiological particle comprises non-template DNA molecules (e.g.,mitochondrial DNA molecules), template DNA molecules, and template RNAmolecules; (c) providing (i) a plurality of transposon end nucleic acidmolecules comprising a transposon end sequence and (ii) a plurality oftransposase nucleic acid molecules; (d) generating a plurality oftemplate DNA fragments in biological particles of the plurality ofbiological particles with the aid of a transposase-nucleic acid complexcomprising a transposase nucleic acid molecule of the plurality oftransposase nucleic acid molecules and a transposon end nucleic acidmolecule of the plurality of transposon end nucleic acid molecules; (e)generating a partition (e.g., a droplet or well) comprising (i) thebiological particle comprising the plurality of template nucleic acidfragments and template RNA molecules; (ii) a plurality of first barcodeoligonucleotide molecules (e.g., first nucleic acid barcode molecules)comprising a barcode sequence; (iii) a plurality of second barcodeoligonucleotide molecules (e.g., second nucleic acid molecules)comprising a barcode sequence and a capture sequence; and (iv) aplurality of reverse transcriptase molecules; (f) generating a barcodedtemplate DNA fragment using a barcode oligonucleotide molecule of theplurality of first barcode oligonucleotide molecules and a template DNAfragment of the plurality of template DNA fragments; and (g) generatinga barcoded cDNA molecule from the template RNA molecules by reversetranscription using a barcode oligonucleotide molecule of the pluralityof second barcode oligonucleotide molecules. In some cases, the blockingagent may comprise bovine serum albumin (BSA). For example, the blockingagent may comprise a solution of 3% BSA in phosphate buffered saline.Lysing the plurality of cells may comprise providing a lysis buffercomprising the cell lysis agent to the plurality of cells. In somecases, the biological particles may be nuclei, which may be isolatedfrom lysed cells by, e.g., washing the lysed cells. In some cases, themethod further comprises generating sequencing reads. Application of themethod may reduce the fraction of sequencing reads deriving frommitochondrial DNA relative to the total number of sequencing readsgenerated. In some cases, the template DNA molecules may comprisechromatin.

In some cases, a method of reducing or removing mitochondrial DNA from asample may comprise providing a sample comprising a plurality of cellscomprising nuclear DNA and mitochondrial DNA; subjecting the pluralityof cells to analysis for accessible chromatin (e.g., tagmentation, asdescribed herein), thereby generating tagmented mitochondrial DNAfragments and tagmented nuclear DNA fragments; and using an enzyme todeplete tagmented mitochondrial DNA fragments, thereby reducing therelative amount of tagmented mitochondrial DNA fragments within thetotal number of fragments. In some cases, the enzyme may be anendonuclease enzyme. For example, the enzyme may be an RNA-guided DNAendonuclease enzyme. The enzyme may be a clustered regularly interspacedshort palindromic (CRISPR) associated protein (e.g., nuclease) such asCas9. The enzyme (e.g., Cas9, such as recombinant Cas9) may be complexedwith one or more guide RNAs (gRNAs). The Cas9-gRNA scheme may be used totarget species for cleavage. For example, tagmented mitochondrial DNAfragments may be cleaved so that mitochondrial-derived DNA fragmentswill not be processed in subsequent sequencing. In this scheme, a gRNAmay be specifically designed to target a species of interest (e.g., atagmented mitochondrial DNA fragment). An enzyme (e.g., Cas9, such asrecombinant Cas9) may be complexed with gRNAs to target loci of humanmitochondrial chromosome. Application of the enzyme-gRNA system mayresult in a reduction in the fraction of mitochondrial DNA fragmentsrelative to a total number of fragments. In an example, a plurality ofgRNAs may be provided to a mixture comprising mitochondrial DNAfragments, where the plurality of gRNAs target different regions ofmitochondrial DNA. For example, the plurality of gRNAs may targetmitochondrial DNA (e.g., human mitochondrial chromosome) about every 250base pairs.

In some cases, the amount of sequencing reads associated withmitochondrial DNA in an ATAC-seq library may be reduced by at leastabout 5%. For example, the amount of sequencing reads associated withmitochondrial DNA may be reduced by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, ormore. In an example the amount of sequencing reads associated withmitochondrial DNA may be reduced by at least about 50%, such as fromabout 30% to about 60%. In some cases, the fraction of sequencing readsassociated with mitochondrial DNA of a plurality of sequencing readsderived from a sample (e.g., a solution, partition, or plurality ofpartitions comprising one or more cells and/or nuclei) may be reduced tobe less than about 60%. For example, the fraction of sequencing readsassociated with mitochondrial DNA may be reduced to less than about 60%,55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of thetotal sequencing reads derived from a sample. In an example, thefraction of sequencing reads associated with mitochondrial DNA may bereduced to between about 1-5% of the total sequencing reads derived froma sample.

In some cases, ATAC-seq and RNA-seq methods may be combined with amethod of reducing non-template (e.g., mitochondria]) fragments. Forexample, a method may comprise generating both barcoded template nucleicacid fragments and barcoded cDNA fragments (e.g., generated via reversetranscription of template RNA molecules) as well as using a blockingagent to reduce non-template fragments (e.g., as described herein).Alternatively or in addition, a method may comprise generating bothbarcoded template nucleic acid fragments and barcoded cDNA fragments(e.g., generated via reverse transcription of template RNA molecules) aswell as using a CRISPR-associated protein to reduce non-templatefragments (e.g., as described herein). In some cases, non-templatefragments may comprise mitochondrial DNA. In some cases, non-templatefragments may comprise mitochondrial RNA. In an example, a biologicalparticle comprising a template DNA molecule, a non-template nucleic acidmolecule comprising ribosomal RNA (rRNA, e.g., mitochondrial rRNA), anda template RNA molecule is provided, and a barcoded cDNA is generatedfrom the template RNA molecule by reverse transcription using a barcodeoligonucleotide (e.g., nucleic acid barcode molecule) (e.g., asdescribed herein). Guide RNA molecules (e.g., sgRNAs) targeted tospecific non-template rRNA (e.g., specific regions of mitochondrialrRNA) or cDNA molecule generated from rRNA molecules may be used incombination with a CRISPR associated protein such as Cas9 to selectivelydeplete sequences associated with rRNA, e.g., upon sequencing aplurality of sequences derived from the biological particle. Therefore,the methods described herein may allow for selective depletion offragments and/or sequences (e.g., sequence reads) associated with anon-template species such as a non-template rRNA and correspondingenhancement of fragments and/or sequences (e.g., sequence reads)associated with template DNA and RNA species.

In some cases, a method for nucleic acid processing comprises: (a)generating a partition (e.g., a droplet or well), wherein the partitioncomprises: (i) a biological particle (e.g., cell); (ii) a plurality ofbarcode oligonucleotide molecules (e.g., nucleic acid barcode molecules)comprising a common barcode sequence; (iii) a plurality of transposonend oligonucleotide molecules (e.g., transposon end nucleic acidmolecules) comprising a transposon end sequence; and (iv) a plurality oftransposase molecules, wherein the biological particle (e.g., cell)comprises a template nucleic acid and a non-template nucleic acid; (b)generating a plurality of template nucleic acid fragments and aplurality of non-template nucleic acid fragments (e.g., mitochondrialnucleic acid fragments) with the aid of a transposase-nucleic acidcomplex comprising a transposase molecule of the plurality oftransposase molecules and a transposon end oligonucleotide molecule ofthe plurality of transposon end oligonucleotide molecules; (c)generating a barcoded template nucleic acid fragment using a barcodeoligonucleotide molecule of the plurality of barcode oligonucleotidemolecules and a template nucleic acid fragment of the plurality oftemplate nucleic acid fragments; and (d) cleaving one or morenon-template nucleic acid fragments of the plurality of non-templatenucleic acid fragments, or derivatives thereof, using (i) one or moreguide ribonucleic acid molecules (gRNAs) targeted to the one or morenon-template nucleic acid fragments, and (ii) a clustered regularlyinterspaced short palindromic (CRISPR) associated (Cas) nuclease. Insome cases, one or more barcoded non-template nucleic acid fragments aregenerated using barcode oligonucleotide molecules of the plurality ofbarcode oligonucleotide molecules and one or more non-template nucleicacid fragments (e.g., mitochondrial nucleic acid fragments) of theplurality of non-template nucleic acid fragments. In some cases, themethod may reduce the amount of fragments and/or barcoded fragmentscomprising and/or deriving from non-template nucleic acids. For example,the method may reduce the total number of non-template nucleic acidfragments and barcoded non-template nucleic acid fragments, e.g., in amixture comprising one or more of template nucleic acid fragments,barcoded template nucleic acid fragments, non-template nucleic acidfragments, and barcoded non-template nucleic acid fragments. In somecases, the endonuclease may be Cas9, such as recombinant Cas9. In somecases, the non-template nucleic acid fragment may comprise amitochondrial DNA fragment. In some cases, the template nucleic acidfragment may comprise a nuclear DNA fragment.

In some cases, a method of generating barcoded nucleic acid fragmentscomprises: (a) providing a plurality of biological particles (e.g.,cells or nuclei), an individual biological particle (e.g., cell ornucleus) comprising a template nucleic acid (e.g., chromatin) and anon-template nucleic acid (e.g., mitochondrial nucleic acid); (b)generating a plurality of template nucleic acid fragments and aplurality of non-template nucleic acid fragments (e.g., mitochondrialnucleic acid fragments) in a biological particle of the plurality ofbiological particles with the aid of a transposase-nucleic acid complexcomprising a transposase nucleic acid molecule and a transposon endnucleic acid molecule; (c) generating a partition (e.g., a droplet orwell) comprising the biological particle comprising the plurality oftemplate nucleic acid fragments and the plurality of non-templatenucleic acid fragments, and a plurality of barcode oligonucleotidemolecules (e.g., nucleic acid barcode molecules) comprising a barcodesequence; (d) generating a barcoded template nucleic acid fragment usinga barcode oligonucleotide molecule of the plurality of barcodeoligonucleotide molecules and a template nucleic acid fragment of theplurality of template nucleic acid fragments; and (e) cleaving one ormore non-template DNA fragments of the plurality of non-template DNAfragments, or derivatives thereof, using (i) one or more guideribonucleic acid molecules (gRNAs) targeted to the one or morenon-template DNA fragments, and (ii) a clustered regularly interspacedshort palindromic (CRISPR) associated (Cas) nuclease. In some cases, oneor more barcoded non-template nucleic acid fragments are generated usingbarcode oligonucleotide molecules of the plurality of barcodeoligonucleotide molecules and one or more non-template nucleic acidfragments (e.g., mitochondrial nucleic acid fragments) of the pluralityof non-template nucleic acid fragments. In some cases, the method mayreduce the amount of fragments and/or barcoded fragments comprisingand/or deriving from non-template nucleic acids. For example, the methodmay reduce the total number of non-template nucleic acid fragments andbarcoded non-template nucleic acid fragments, e.g., in a mixturecomprising one or more of template nucleic acid fragments, barcodedtemplate nucleic acid fragments, non-template nucleic acid fragments,and barcoded non-template nucleic acid fragments. In some cases, theendonuclease may be Cas9, such as recombinant Cas9. In some cases, thenon-template nucleic acid fragment may comprise a mitochondrial DNAfragment. In some cases, the template nucleic acid fragment may comprisechromatin. In some cases, a method of generating barcoded nucleic acidfragments comprises: (a) generating a partition (e.g., a droplet orwell), wherein the partition comprises: (i) a biological particle (e.g.,cell or nucleus), wherein the biological particle comprises template DNAmolecules (e.g., chromatin), non-template DNA molecules (e.g.,mitochondrial DNA molecules), and template RNA molecules; (ii) aplurality of first barcode oligonucleotide molecules (e.g., firstnucleic acid barcode molecules) comprising a barcode sequence; (iii) aplurality of transposon end nucleic acid molecules comprising atransposon end sequence; (iv) a plurality of transposase nucleic acidmolecules; (v) a plurality of second barcode oligonucleotide molecules(e.g., second nucleic acid barcode molecules) comprising a barcodesequence and a capture sequence; and (vi) a plurality of reversetranscriptase molecules; (b) generating a plurality of template DNAfragments and a plurality of non-template DNA fragments (e.g.,mitochondrial nucleic acid fragments) with the aid of atransposase-nucleic acid complex comprising a transposase nucleic acidmolecule of the plurality of transposase nucleic acid molecules and atransposon end nucleic acid molecule of the plurality of transposon endnucleic acid molecules; (c) generating a barcoded template DNA fragmentusing a barcode oligonucleotide molecule of the plurality of firstbarcode oligonucleotide molecules and a template DNA fragment of theplurality of template DNA fragments; (d) generating a barcoded cDNAmolecule from the template RNA molecules by reverse transcription usinga barcode oligonucleotide molecule of the plurality of second barcodeoligonucleotide molecules; and (e) cleaving one or more non-template DNAfragments of the plurality of non-template DNA fragments, or derivativesthereof, using (i) one or more guide ribonucleic acid molecules (gRNAs)targeted to the one or more non-template DNA fragments, and (ii) aclustered regularly interspaced short palindromic (CRISPR) associated(Cas) nuclease. In some cases, one or more barcoded non-template DNAfragments are generated using barcode oligonucleotide molecules of theplurality of barcode oligonucleotide molecules and one or morenon-template DNA fragments (e.g., mitochondrial DNA fragments) of theplurality of non-template DNA fragments. In some cases, the method mayreduce the amount of fragments and/or barcoded fragments comprisingand/or deriving from non-template DNA. For example, the method mayreduce the total number of non-template DNA fragments and barcodednon-template DNA fragments, e.g., in a mixture comprising one or more oftemplate DNA fragments, barcoded template DNA fragments, non-templateDNA fragments, and barcoded non-template DNA fragments. In some cases,the endonuclease may be Cas9, such as recombinant Cas9. In some cases,the non-template DNA fragment may comprise a mitochondrial DNA fragment.In some cases, the template DNA fragment may comprise chromatin.

In some cases, a method of generating barcoded nucleic acid fragmentscomprises: (a) providing a biological particle (e.g., cell) comprisingtemplate DNA molecules, non-template DNA molecules, and template RNAmolecules; (b) generating a plurality of template DNA fragments andnon-template DNA fragments in the biological particle with the aid of atransposase-nucleic acid complex comprising a transposase nucleic acidmolecule and a transposon end nucleic acid molecule; (c) generating apartition (e.g., a droplet or well) comprising (i) the biologicalparticle comprising template DNA fragments, non-template DNA fragments,and template RNA molecules; (ii) a plurality of first barcodeoligonucleotide molecules (e.g., first nucleic acid barcode molecules)comprising a common barcode sequence; (iii) a plurality of secondbarcode oligonucleotide molecules (e.g., second nucleic acid molecules)comprising a barcode sequence and a capture sequence; and (iv) aplurality of reverse transcriptase molecules; (d) generating a barcodedtemplate DNA fragment using a barcode oligonucleotide molecule of theplurality of first barcode oligonucleotide molecules and a template DNAfragment of the plurality of template DNA fragments; (e) generating abarcoded cDNA molecule from the template RNA molecules by reversetranscription using a barcode oligonucleotide molecule of the pluralityof second barcode oligonucleotide molecules; and (f) cleaving one ormore non-template DNA fragments of the plurality of non-template DNAfragments, or derivatives thereof, using (i) one or more guideribonucleic acid molecules (gRNAs) targeted to the one or morenon-template nucleic acid fragments, and (ii) a clustered regularlyinterspaced short palindromic (CRISPR) associated (Cas) nuclease. Insome cases, one or more barcoded non-template DNA fragments aregenerated using barcode oligonucleotide molecules of the plurality ofbarcode oligonucleotide molecules and one or more non-template DNAfragments (e.g., mitochondrial DNA fragments) of the plurality ofnon-template DNA fragments. In some cases, the method may reduce theamount of fragments and/or barcoded fragments comprising and/or derivingfrom non-template DNA. For example, the method may reduce the totalnumber of non-template DNA fragments and barcoded non-template DNAfragments, e.g., in a mixture comprising one or more of template DNAfragments, barcoded template DNA fragments, non-template DNA fragments,and barcoded non-template DNA fragments. In some cases, the endonucleasemay be Cas9, such as recombinant Cas9. In some cases, the non-templateDNA fragment may comprise a mitochondrial DNA fragment. In some cases,the template DNA fragment may comprise a nuclear DNA fragment.

In some cases, a method of generating barcoded nucleic acid fragmentscomprises: (a) providing a partition (e.g., a droplet or well), whereinthe partition comprises (i) a biological particle (e.g., cell ornucleus), (ii) a single solid or semi-solid particle (e.g., bead, suchas a gel bead), and (iii) a plurality of transposase nucleic acidmolecules, wherein the single biological particle comprises a templatenucleic acid molecule and a non-template nucleic acid molecule andwherein the single solid or semi-solid particle (e.g., bead) comprises abarcoded oligonucleotide (e.g., nucleic acid barcode molecule)releasably coupled thereto; (b) subjecting the partition to conditionssufficient to release the template nucleic acid molecule and thenon-template nucleic acid molecule from the single biological particle;(c) subjecting the partition to conditions sufficient to release thebarcoded oligonucleotide from the solid or semi-solid particle; (d)subjecting the partition to conditions sufficient to cause transpositionof the barcoded oligonucleotides into the template nucleic acid moleculeand the non-template nucleic acid molecule with the aid of a transposomecomplex generated from at least a subset of the plurality of transposasenucleic acid molecules; (e) fragmenting (i) the template nucleic acidmolecule into a plurality of double-stranded template nucleic acidfragments comprising the barcoded oligonucleotides and (ii) thenon-template nucleic acid molecule into a plurality of double-strandednon-template nucleic acid fragments comprising the barcodedoligonucleotides; and (f) cleaving one or more non-template nucleic acidfragments of the plurality of non-template nucleic acid fragments, orderivatives thereof, using (i) one or more guide ribonucleic acidmolecules (gRNAs) targeted to the one or more non-template nucleic acidfragments, and (ii) a clustered regularly interspaced short palindromic(CRISPR) associated (Cas) nuclease. In some cases, the method may reducethe amount of fragments comprising and/or deriving from non-templatenucleic acids. For example, the method may reduce the total number ofnon-template nucleic acid fragments, e.g., in a mixture comprising oneor more of template nucleic acid fragments, barcoded template nucleicacid fragments, non-template nucleic acid fragments, and barcodednon-template nucleic acid fragments. In some cases, the endonuclease maybe Cas9, such as recombinant Cas9. In some cases, the non-templatenucleic acid fragment may comprise a mitochondrial DNA fragment. In somecases, the template nucleic acid fragment may comprise chromatin.

The present methods may have the effect of reducing or removingnon-template nucleic acids (e.g., mitochondrial DNA) and/or enhancingaccessible chromatin. The methods may be used with any cell type (e.g.,as described herein). For example, the methods may be used with a humanor a mammalian cell. The methods may also be used with any concentrationof cells. For example, the methods of reducing or removing non-templatenucleic acids (e.g., mitochondrial DNA) and/or enhancing accessiblechromatin may be used with an isolated cell. In another example, themethod of reducing or removing non-template nucleic acids (e.g.,mitochondrial DNA) and/or enhancing accessible chromatin may be usedwith a plurality of cells, such as a plurality of cells within aplurality of droplets or a plurality of cells in a bulk solution. Themethod of reducing or removing non-template nucleic acids (e.g.,mitochondrial DNA) and/or enhancing accessible chromatin may be appliedto any library type that requires a reducing in sequencing readsassociated with non-template nucleic acids (e.g., mitochondrial DNA) orRNA, including RNA-seq.

A method of reducing non-template (e.g., mitochondrial) reads and/orenhancing reads associated with accessible chromatin relative to a totalnumber of reads associated with a sample (e.g., a cell or a plurality ofcells) may be applied to any of the methods described herein. Forexample, a blocking agent (e.g., BSA) may be provided to any sampledescribed herein. As described above, use of a blocking agent may reduceor prevent non-specific adsorption of mitochondrial DNA to nuclei suchthat mitochondrial DNA can be washed away during nuclei isolation.Similarly, an endonuclease may be provided after any tagmentationprocess described herein to reduce the relative fraction of tagmentedmitochondrial DNA in a sample comprising tagmented DNA.

Additional details and methods regarding processing nucleic acid samplesand reducing non-template (e.g., mitochondrial) reads can be found in,for example: Gu et al. (Gu, W.; Crawford, E. D.; O'Donovan, B. D.,Wilson, M. R., Chow, E. D., Retallack, H., and DeRisi, J. L. “Depletionof Abundant Sequences by Hybridization (DASH): using Cas9 to removeunwanted high-abundance species in sequencing libraries and molecularcounting applications,” Genome Biology (2016) 17:41), Montefiori et al.(Montefiori, L.; Hernandez, L.; Zhang, Z.; Gilad, Y.; Ober, C.;Crawford, G.; Nobrega, M.; and Sakabe, N. J. “Reducing mitochondrialreads in ATAC-seq using CRISPR/Cas9,” Scientific Reports (2017) 7:2451),and Corces et al. (Corces, M. R.; Trevino, A. E.; Hamilton, E. G.;Greenside, P. G.; Sinnott-Armstrong, N. A.; Vesuna, S.; Satpathy, A. T.;Rubin, A. J.; Montine, K. S.; Wu, B.; Kathiria, A.; Cho, S. W.; Mumbach,M. R.; Carter, A. C.; Kasowski, M.; Orloff, L. A.; Risca, V. I.;Kundaje, A.; Khavari, P. A.; Montine, T. J.; Greenleaf, W. J.; andChang, H. Y. “An improved ATAC-seq protocol reduces background andenables interrogation of frozen tissues,” Nature Methods (2017)), eachof which is herein incorporated by reference in its entirety.

Kits

Also provided herein are kits for analyzing the accessible chromatin(e.g., for ATAC-seq) and/or RNA transcripts of individual cells or smallpopulations of cells. The kits may include one or more of the following:one, two, three, four, five or more, up to all of partitioning fluids,including both aqueous buffers and non-aqueous partitioning fluids oroils, nucleic acid barcode libraries that are releasably associated withbeads, as described herein, microfluidic devices, reagents fordisrupting cells amplifying nucleic acids, and providing additionalfunctional sequences on fragments of cellular nucleic acids orreplicates thereof, as well as instructions for using any of theforegoing in the methods described herein.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 9 shows a computer system 901that is programmed or otherwise configured to, e.g., (i) control amicrofluidics system (e.g., fluid flow), (ii) sort occupied dropletsfrom unoccupied droplets, (iii) polymerize droplets, (iv) performsequencing applications, (v) generate and maintain a library of DNA orcDNA fragments, and/or (vi) analyze areas of accessible chromatin. Thecomputer system 901 can regulate various aspects of the presentdisclosure, such as, for example, e.g., regulating fluid flow rate inone or more channels in a microfluidic structure, regulatingpolymerization application units, regulating conditions for certainreactions described herein. The computer system 901 can be an electronicdevice of a user or a computer system that is remotely located withrespect to the electronic device. The electronic device can be a mobileelectronic device.

The computer system 901 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 905, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 901 also includes memory or memorylocation 910 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 915 (e.g., hard disk), communicationinterface 920 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 925, such as cache, other memory,data storage and/or electronic display adapters. The memory 910, storageunit 915, interface 920 and peripheral devices 925 are in communicationwith the CPU 905 through a communication bus (solid lines), such as amotherboard. The storage unit 915 can be a data storage unit (or datarepository) for storing data. The computer system 901 can be operativelycoupled to a computer network (“network”) 930 with the aid of thecommunication interface 920. The network 930 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 930 in some cases is atelecommunication and/or data network. The network 930 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 930, in some cases with the aid of thecomputer system 901, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 901 to behave as a clientor a server.

The CPU 905 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 910. The instructionscan be directed to the CPU 905, which can subsequently program orotherwise configure the CPU 905 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 905 can includefetch, decode, execute, and writeback.

The CPU 905 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 901 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 915 can store files, such as drivers, libraries andsaved programs. The storage unit 915 can store user data, e.g., userpreferences and user programs. The computer system 901 in some cases caninclude one or more additional data storage units that are external tothe computer system 901, such as located on a remote server that is incommunication with the computer system 901 through an intranet or theInternet.

The computer system 901 can communicate with one or more remote computersystems through the network 930. For instance, the computer system 901can communicate with a remote computer system of a user (e.g.,operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 901 via the network 930.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 901, such as, for example, on the memory910 or electronic storage unit 915. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 905. In some cases, the code canbe retrieved from the storage unit 915 and stored on the memory 910 forready access by the processor 905. In some situations, the electronicstorage unit 915 can be precluded, and machine-executable instructionsare stored on memory 910.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 901, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 901 can include or be in communication with anelectronic display 935 that comprises a user interface (UI) 940 forproviding, for example, results of sequencing analysis, correlatingsequencing reads to areas of accessible chromatin, etc. Examples of UIsinclude, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 905. Thealgorithm can, for example, perform sequencing reactions, correlatingsequencing reads to areas of accessible chromatin, etc.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

EXAMPLES

Although one or more of the Examples herein make use of partitions thatcomprise droplets in an emulsion (e.g., droplet emulsion partition), anyof the above-described partitions (such as wells) can be utilized in themethods, systems, and compositions described below.

Example 1 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Forked Adaptors Comprising Transposon End Sequences

A plurality of transposase molecules, a plurality of cells of interest(or a plurality of nuclei harvested from cells of interest, e.g., bynonionic detergents such as NP-40 (IGEPAL CA-630) or Triton X-100), anda plurality of barcode oligonucleotides (e.g., nucleic acid barcodemolecules) are partitioned such that at least some partitions (e.g.,droplets or wells) comprise a plurality of transposase molecules, asingle cell (or nucleus), and a plurality of barcode oligonucleotidescomprising a sequencing primer sequence, a barcode sequence, and atransposon end sequence. In some embodiments, the plurality of barcodeoligonucleotides are attached to a solid or semi-solid particle such asa gel bead and partitioned such that at least some partitions (e.g.,droplets or wells) comprise transposase molecules, a single cell (ornucleus), and support (e.g., a single gel bead).

For example, in some embodiments, aqueous droplet emulsion partitionsare generated as described herein such that at least some of thedroplets formed will comprise transposase molecules, cell lysisreagents, a single cell, and a single bead (e.g., gel bead) comprising aplurality of barcoded forked adapter oligonucleotides. See, e.g., FIG.10 . The cells are then lysed within the droplets in a manner thatreleases template nucleic acid molecules from cell nuclei into theirrespective droplets, but that substantially maintains native chromatinorganization. Droplets are then generally processed as outlined in FIG.11A.

Specifically, a droplet is subjected to conditions such that thebarcoded forked adaptor oligonucleotides are released from the bead(e.g., gel bead) into the aqueous droplet (e.g., by depolymerization ordegradation of the bead using a reducing agent, such as DTT). Althoughthe forked adaptors can be prepared in a variety of differentconfigurations, an exemplary forked adaptor is illustrated in FIG. 12Aand shows a partially complementary double-stranded oligonucleotidecomprising a first oligonucleotide strand releasably attached to a bead(e.g., gel bead) and a second partially complementary oligonucleotidestrand. With continued reference to FIG. 12A, the first strand comprisesa transposon end sequence (“mosaic end” or “ME”), a barcode sequence(“BC”), and a sequencing primer sequence (“R1”). The partiallycomplementary second strand comprises: (i) a region fully complementaryto the transposon end sequence (“ME”); (ii) a region fully complementaryto the barcode sequence (“BC,”); and (iii) a primer sequence (“R2”)partially complementary to the first strand primer sequence.

In alternative embodiments (e.g., FIG. 12B), the double-stranded forkedadaptor of FIG. 12A further comprises: (a) a first oligonucleotidestrand further comprising a P5 sequence releasably attached to the gelbead; and (b) a second partially complementary oligonucleotide strandfurther comprising an index sequence (“i7”) and a P7 sequence.

After forked adaptors are released from a bead (e.g., gel bead) into adroplet, the droplet is subjected to conditions such that atransposase-nucleic acid complex is formed comprising a transposasemolecule and two forked adaptors. See FIG. 11A, step 2. The droplets arethen subjected to conditions such that the transposase-nucleic acidcomplexes integrate the transposon end sequences into the templatenucleic acid and fragment the template nucleic acid into double-strandedtemplate nucleic acid fragments flanked by the forked adaptors. See FIG.11A, step 3. In alternative embodiments, cells (or nuclei) arepermeabilized/permeable and the transposase-nucleic acid complexes enterthe nucleus to fragment the template nucleic acid. Cells are then lysedto release double-stranded template nucleic acid fragments. Because thetransposase-nucleic acid complex can only act on a nucleosome-freetemplate, the double-stranded template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to fill any gaps created fromthe transposition reaction and to generate a library suitable for nextgeneration high throughput sequencing (e.g., subjecting the fragments,or derivatives thereof, to one or more reactions (e.g., nucleic acidamplification) to add functional sequences to facilitate Illuminasequencing; see FIG. 11B). The fully constructed library is thensequenced according to any suitable sequencing protocol.

Example 2 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Forked Adaptors and Transposase-Nucleic Acid Complexes

A plurality of transposase-nucleic acid complexes, a plurality of cellsof interest (or a plurality of nuclei harvested from cells of interest),and a plurality of barcode oligonucleotides are partitioned such that atleast some partitions (e.g., droplets or wells) comprise a plurality oftransposase-nucleic acid complexes, a single cell (or nucleus), and aplurality of barcode oligonucleotides (e.g., nucleic acid barcodemolecules) comprising a sequencing primer sequence and a barcodesequence. In some embodiments, the plurality of barcode oligonucleotidesare attached to a solid or semi-solid particle (e.g., a bead, such as agel bead) and partitioned such that at least some partitions (e.g.,droplets or wells) comprise transposase-nucleic acid complexes, a singlecell (or nucleus), and a single solid or semi-solid particle (e.g., gelbead). In alternative embodiments, a plurality of transposase moleculesand a plurality of transposon end sequence oligonucleotides arepartitioned along with a single cell (or nucleus) and the barcodeoligonucleotides and transposase-nucleic acid complexes are generated inthe partition (e.g., droplet or well).

For example, in some embodiments, droplet emulsion partitions aregenerated as described herein such that at least some of the dropletscomprise transposase-nucleic acid complexes, cell lysis reagents, T4 DNAligase, a single cell or cell nucleus, and a single bead (e.g., gelbead) comprising a plurality of barcoded forked adapteroligonucleotides. See, e.g., FIG. 13 .

An individual transposase-nucleic acid complex comprises a transposaseand a pair of double-stranded oligonucleotides each comprising atransposon end sequence (e.g., an ME sequence). See FIGS. 13-15 . Insome embodiments, the double-stranded transposon-end sequence containingoligonucleotides further comprise a spacer sequence.

After droplet formation, single cells, if present, are lysed in a mannerthat releases template nucleic acid molecules from the nucleus into thedroplet, but that substantially maintains native chromatin organization.Droplets are then generally processed as outlined in FIG. 14A.

Specifically, a droplet is subjected to conditions such that the forkedadaptors are released from the bead (e.g., gel bead) into the aqueousdroplet (e.g., by depolymerization or degradation of the bead using areducing agent, such as DTT). See FIG. 14A, step 1 a. Although theforked adaptors can be prepared in a variety of differentconfigurations, an exemplary forked adaptor is illustrated in FIG. 15Aand shows a double-stranded oligonucleotide comprising a firstoligonucleotide strand releasably attached to a bead (e.g., gel bead)and a second partially complementary oligonucleotide strand. Withcontinued reference to FIG. 15A, the first strand comprises a barcodesequence (“BC”), a primer sequence (“R1”), and a transposon end sequence(“mosaic end” or “ME”). The partially complementary second strandcomprises a region fully complementary to the barcode sequence (“BC”), aprimer sequence (“R2”) partially complementary to the first strandprimer sequence, and a region fully complementary to the transposon endsequence (“ME”). In some embodiments, the first strand further comprisesa phosphorothioate linkage in the terminal nucleotide at the 3′ end. Inother embodiments, the first strand comprises phosphorothioate linkagesin the last 3-5 nucleotides at the 3′ end. In still other embodiments,the first strand comprises phosphorothioate linkages throughout thefirst strand. The first oligonucleotide strand may further comprise a P5adapter sequence releasably attached to the bead (e.g., gel bead). Thesecond partially complementary oligonucleotide strand may furthercomprise an index primer (“i7”) and an adaptor sequence different thanthe first strand (“P7”). Similarly, FIG. 15B shows a double-strandedoligonucleotide comprising a first oligonucleotide strand releasablyattached to a bead (e.g., gel bead) and a second partially complementaryoligonucleotide strand. With continued reference to FIG. 15B, the firststrand comprises a barcode sequence (“BC”) and a primer sequence (“R1”).The partially complementary second strand comprises a region fullycomplementary to the barcode sequence (“BC”) and a primer sequence(“R2”) partially complementary to the first strand primer sequence. Insome embodiments, the first strand further comprises a phosphorothioatelinkage in the terminal nucleotide at the 3′ end. In other embodiments,the first strand comprises phosphorothioate linkages in the last 3-5nucleotides at the 3′ end. In still other embodiments, the first strandcomprises phosphorothioate linkages throughout the first strand. Thefirst oligonucleotide strand may further comprise a P5 adapter sequencereleasably attached to the bead (e.g., gel bead). The second partiallycomplementary oligonucleotide strand may further comprise an indexprimer (“i7”) and an adaptor sequence different than the first strand(“P7”).

After forked adaptors are released from a bead (e.g., gel bead) into adroplet, the droplet is subjected to conditions such that thetransposase-nucleic acid complexes integrate the transposon endsequences into the template nucleic acid and fragment the templatenucleic acid into double-stranded template nucleic acid fragmentsflanked by transposon end sequences. See FIG. 14A, step 1 b. Inalternative embodiments, cells (or nuclei) are permeabilized/permeableand the transposase-nucleic acid complexes enter the nuclei to fragmentthe template nucleic acids therein. Cells, if present, are then lysed torelease the double-stranded template nucleic acid fragments. Because thetransposase-nucleic acid complex can only act on a nucleosome-freetemplate, the double-stranded template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus. The forked adaptors are then ligated onto the ends ofthe double-stranded stranded template nucleic acid fragments. See FIG.14A, step 2.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to fill any gaps created fromthe transposition reaction and to generate a library suitable for nextgeneration high throughput sequencing (e.g., subjecting the fragments,or derivatives thereof, to one or more reactions (e.g., nucleic acidamplification) to add functional sequences to facilitate Illuminasequencing; see FIG. 14B). The fully constructed library is thensequenced according to any suitable sequencing protocol. In someembodiments, custom sequencing primers directed against the spacer-MEsequence are utilized to avoid sequencing the barcode-spacer-ME regionof the library.

Example 3 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Adaptors Comprising a T7 Promoter Sequence

A plurality of transposase molecules, a plurality of cells of interest(or a plurality of nuclei harvested from cells of interest), and aplurality of barcode oligonucleotides are partitioned such that at leastsome partitions (e.g., droplets or wells) comprise a plurality oftransposase molecules, a single cell (or nucleus), and a plurality ofbarcode oligonucleotides (e.g., nucleic acid barcode molecules)comprising a T7 promoter sequence, a sequencing primer sequence, abarcode sequence, and a transposon end sequence. In some embodiments,the plurality of barcode oligonucleotides are attached to a solid orsemi-solid particle (e.g., a bead, such as a gel bead) and partitionedsuch that at least some partitions (e.g., droplets or wells) comprisetransposase molecules, a single cell (or nucleus), and a single solid orsemi-solid particle (e.g., gel bead).

For example, in some embodiments, droplet emulsion partitions aregenerated as previously described such that at least some droplets(e.g., aqueous droplets) that comprise transposase molecules, cell lysisreagents, a single cell (or nucleus), and a single bead (e.g., gel bead)comprising partially double-stranded T7 promoter oligonucleotideadaptors. See, e.g., FIG. 16 . The cells, if present, are then lysed ina manner that releases template nucleic acid molecules from the nucleusinto the droplet, but that substantially maintains native chromatinorganization. Droplets are then generally processed as outlined in FIG.17 .

Specifically, a droplet (e.g., an aqueous droplet) is subjected toconditions such that the partially double-stranded adaptors are releasedfrom the gel bead into the droplet (e.g., by depolymerization ordegradation of the bead using a reducing agent, such as DTT). See FIG.17 , step 1. Although the partially double-stranded adaptors can beprepared in a variety of different configurations, an exemplarypartially double-stranded adaptor is illustrated in FIG. 18 and shows apartially double-stranded oligonucleotide comprising a firstoligonucleotide strand releasably attached to a bead (e.g., a gel bead)and a second, shorter complementary oligonucleotide strand. Withcontinued reference to FIG. 18 , the first strand comprises a transposonend (“mosaic end” or “ME”) sequence, a barcode sequence (“BC”), apartial sequencing primer sequence (“pR1” or “Partial R1”), and a T7promoter sequence while the second oligonucleotide strand comprises asequence fully complementary to the transposon end sequence (“ME”).

After partially double-stranded adaptors are released from a bead (e.g.,gel bead) into a droplet, the droplet is subjected to conditions suchthat a transposase-nucleic acid complex is formed comprising atransposase molecule and two partially double-stranded oligonucleotidesadaptors. See FIG. 17 , step 2. The droplet or plurality of droplets isthen subjected to conditions such that the transposase-nucleic acidcomplexes integrate the adaptors into the template nucleic acid andproduce double-stranded template nucleic acid fragments flanked by thepartially double-stranded adaptors. See FIG. 17 , step 3. In alternativeembodiments, cells (or nuclei) are permeabilized/permeable and thetransposase-nucleic acid complexes enter the nuclei to fragment thetemplate nucleic acids therein. Cells, if present, are then lysed torelease the double-stranded template nucleic acid fragments. Because thetransposase-nucleic acid complex can only act on a nucleosome-freetemplate, the double-stranded template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to fill any gaps created fromthe transposition reaction. RNA is generated from the double-strandedtemplate nucleic acid fragments using an in vitro transcription reactionand T7 RNA polymerase. RNA is collected and purified, followed by firstand second strand cDNA synthesis. Double-stranded cDNA molecules arethen further processed (including fragmentation and adaptor insertionby, e.g., a second transposase-mediated fragmentation) to generate alibrary suitable for next generation high throughput sequencing (e.g.,subjecting the fragments, or derivatives thereof, to one or morereactions (e.g., nucleic acid amplification) to add functional sequencesto facilitate Illumina sequencing). The fully constructed library isthen sequenced according to any suitable sequencing protocol.

Example 4 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Transposition of Sequencing Adaptors Followed by RandomPriming and Extension

A plurality of transposase molecules, a plurality of cells of interest(or a plurality of nuclei harvested from cells of interest), and aplurality of barcode oligonucleotides are partitioned such that at leastsome partitions (e.g., droplets or wells) comprise a plurality oftransposase molecules, a single cell (or nucleus), and a plurality ofbarcode oligonucleotides (e.g., nucleic acid barcode molecules)comprising a sequencing primer sequence, a barcode sequence, and atransposon end sequence. In some embodiments, the plurality of barcodeoligonucleotides are attached to a solid or semi-solid particle (e.g., abead, such as a gel bead) and partitioned such that at least somepartitions (e.g., droplets or wells) comprise transposase molecules, asingle cell (or nucleus), and a single solid or semi-solid particle(e.g., gel bead).

For example, in some embodiments, droplet emulsion partitions aregenerated as previously described such that at least some droplets thatcomprise transposase molecules, cell lysis reagents, a single cell (ornucleus), and a single bead (e.g., gel bead) comprising partiallydouble-stranded barcoded oligonucleotide adaptors. See, e.g., FIG. 19 .The cells, if present, are then lysed in a manner that releases templatenucleic acid molecules from the nucleus into the aqueous droplet, butthat substantially maintains native chromatin organization. Droplets arethen generally processed as outlined in FIG. 20 .

Specifically, a droplet (e.g., an aqueous droplet) is subjected toconditions such that partially double-stranded adaptors are releasedfrom a bead (e.g., gel bead) into the droplet (e.g., by depolymerizationor degradation of the bead using a reducing agent, such as DTT). SeeFIG. 20 , step 1. Although the partially double-stranded adaptors can beprepared in a variety of different configurations, an exemplarypartially double-stranded adaptor is illustrated in FIG. 21 and shows apartially double-stranded oligonucleotide comprising a firstoligonucleotide strand releasably attached to a bead (e.g., gel bead)and a second, shorter complementary oligonucleotide strand. Withcontinued reference to FIG. 21 , the first strand comprises a transposonend (“mosaic end” or “ME”) sequence, a barcode sequence (“BC”), and apartial sequencing primer sequence (“pR1” or “Partial R1”) while thesecond oligonucleotide strand comprises a sequence fully complementaryto the transposon end sequence (“MErc”).

After partially double-stranded adaptors are released from a bead (e.g.,gel bead) into a droplet (e.g., an aqueous droplet), the droplet issubjected to conditions such that a transposase-nucleic acid complex isformed comprising a transposase molecule and two partiallydouble-stranded oligonucleotides. See FIG. 20 , step 2. The droplets arethen subjected to conditions such that the transposase-nucleic acidcomplexes integrate the adaptors into the template nucleic acid andproduce double-stranded template nucleic acid fragments flanked by thepartially double-stranded adaptors. See FIG. 20 , step 3. In alternativeembodiments, cells (or nuclei) are permeabilized/permeable and thetransposase-nucleic acid complexes enter the nucleus to fragment thetemplate nucleic acid. Cells, if present, are then lysed to release thedouble-stranded template nucleic acid fragments. Because thetransposase-nucleic acid complex can only act on a nucleosome-freetemplate, the double-stranded template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to generate a library suitablefor next generation high throughput sequencing. In some embodiments, forexample, double-stranded template nucleic acid fragments are processedin bulk in a random priming extension reaction. See, e.g., FIG. 22 . Therandom extension primer has a sequence of random nucleotides (N-mer)and, for example, can be attached to second PCR handle (e.g., partial R2sequence, see FIG. 22 ). The random extension primers are annealed tothe double-stranded template nucleic acid fragments, or derivativesthereof, and extended (e.g., FIG. 22 ). Reactions can then be cleaned-upand extension products subjected to one or more reactions (e.g., nucleicacid amplification) to add functional sequences to facilitate Illuminasequencing (e.g., FIG. 22 ). The fully constructed library is thensequenced according to any suitable sequencing protocol.

Example 5 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Transposase-Nucleic Acid Complexes and Barcoded Adaptors

In some embodiments, artificial transposons are designed to insertsequences of interest into a target DNA molecule (e.g., open chromatin).For example, an artificial transposon oligonucleotide comprising abarcode sequence and an adapter sequence is flanked by a transposon endsequence on each end of the oligonucleotide (see, e.g., FIG. 23 ). Aplurality of transposase molecules, a plurality of cells of interest (ora plurality of nuclei harvested from cells of interest), and a pluralityof artificial transposon oligonucleotides are partitioned such that atleast some partitions (e.g., droplets or wells) comprise a plurality ofartificial transposon oligonucleotides, a plurality of transposasemolecules, and a single cell (or nucleus). In some embodiments, theplurality of artificial transposon oligonucleotides are attached to asolid or semi-solid particle (e.g., bead, such as a gel bead) andpartitioned such that at least some partitions (e.g., droplets or wells)comprise a plurality of transposase molecules, a single cell (ornucleus), and a single solid or semi-solid particle (e.g., gel bead). Inother embodiments, a plurality of transposon nucleic acid complexescomprising an artificial transposon oligonucleotide comprising a barcodesequence and an adapter sequence flanked by transposon end sequenced arepartitioned such that at least some partitions comprise a plurality oftransposon nucleic acid complexes and a single cell (or nucleus). Insome embodiments, the plurality of artificial transposonoligonucleotides are attached to a solid or semi-solid particle (e.g.,bead, such as a gel bead) and partitioned such that at least somepartitions (e.g., droplets or wells) comprise a single cell (or nucleus)and a single solid or semi-solid particle (e.g., gel bead).

For example, in some embodiments, droplet emulsion partitions aregenerated as previously described such that at least some droplets thatcomprise transposase molecules, cell lysis reagents, a single cell (ornucleus), and a single bead (e.g., gel bead). The cells are then lysedto release template nucleic acid molecules from the nucleus into theaqueous droplet. Droplets are then generally processed as outlined inFIG. 23 .

Specifically, a droplet (e.g., aqueous droplet) is subjected toconditions such that barcoded adaptors are released from a bead (e.g.,gel bead) into the droplet (e.g., by depolymerization or degradation ofthe bead using a reducing agent, such as DTT). Although the barcodedadaptors can be prepared in a variety of different configurations, anexemplary barcoded adaptor is illustrated in FIG. 23 and is adouble-stranded oligonucleotide releasably attached to a bead (e.g., gelbead), wherein the barcoded adaptor comprises a pair of transposon end(“mosaic end” or “ME”) sequences flanking a barcode sequence (“BC”) andan adaptor sequence (“P5”).

After barcoded adaptors are released from a bead (e.g., gel bead) into adroplet, the droplet is subjected to conditions such that atransposase-nucleic acid complex is formed comprising a transposasemolecule and a barcoded adaptor comprising a pair of transposon endsequences. See FIG. 23 . The droplet or plurality of droplets is thensubjected to conditions such that the transposase-nucleic acid complexesintegrate the barcoded adaptors into the template nucleic acid. See FIG.23 . In alternative embodiments, cells (or nuclei) arepermeabilized/permeable and the transposase-nucleic acid complexes enterthe nuclei to perform the transposition reaction. Cells, if present, arethen lysed to release the transposon-containing template nucleic acidfragments.

The barcode-transposed template nucleic acids are then collected fromthe droplets and processed in bulk to fragment the barcode-transposedtemplate nucleic acids and to generate a library suitable for nextgeneration high throughput sequencing (e.g., subjecting the fragments,or derivatives thereof, to one or more reactions (e.g., nucleic acidamplification) to add functional sequences to facilitate Illuminasequencing). The fully constructed library is then sequenced accordingto any suitable sequencing protocol.

Example 6 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Gel Bead-Functionalized Transposase-Nucleic Acid Complexes

A plurality of transposase nucleic acid complexes and a plurality ofcells of interest (or a plurality of nuclei harvested from cells ofinterest) are partitioned such that at least some partitions (e.g.,droplets or wells) comprise a single cell (or nucleus) and a pluralityof transposase nucleic acid complexes comprising a transposase moleculeand a barcode oligonucleotide (e.g., nucleic acid barcode molecule)comprising a sequencing primer sequence, a barcode sequence, and atransposon end sequence (see, e.g., FIGS. 24A-24B and 25A-25B). In someembodiments, the plurality of transposase nucleic acid complexes areattached to a solid or semi-solid particle (e.g., bead, such as a gelbead) (see, e.g., FIGS. 24A-24B and 26 ) and partitioned such that atleast some partitions (e.g., droplets or wells) comprise a single cell(or nucleus) and a single solid or semi-solid particle (e.g., gel bead).

For example, in some embodiments, droplet emulsion partitions aregenerated as previously described such that at least some droplets(e.g., aqueous droplets) comprise cell lysis reagents, a single cell (ornucleus), and a single bead (e.g., gel bead) functionalized with atransposase-nucleic acid complex. The cells are then lysed in a mannerthat releases template nucleic acid molecules from the nuclei into therespective droplets, but that substantially maintains native chromatinorganization.

Droplets (e.g., aqueous droplets) are then subjected to conditions suchthat the transposase-nucleic acid complexes are released from beads(e.g., gel beads) into the respective droplets (e.g., bydepolymerization or degradation of beads using a reducing agent, such asDTT). Although the transposase-nucleic acid complexes can be prepared ina variety of different configurations, a transposase-nucleic acidcomplex is illustrated in FIG. 24A and shows a complex comprising atransposase, a first partially double-stranded oligonucleotide, and asecond partially double-stranded oligonucleotide. Continuing with theembodiment of FIG. 24A, the first partially double-strandedoligonucleotide comprises: (a) a first strand releasably attached to abead (e.g., gel bead), wherein the first strand comprises a transposonend sequence (“ME”), a barcode sequence (“BC”), and a first sequencingprimer sequence (“R1”); and (b) a second strand complementary to thetransposon end sequence of the first oligonucleotide strand (“ME”). Withcontinued reference to FIG. 24A, the second partially double-strandedoligonucleotide comprises: (a) a first oligonucleotide strand comprisinga transposon end sequence (“ME”) and a second primer sequence (“R2”);and (b) a second strand complementary to the transposon end sequence(“MiE_(rc)”). See also FIG. 24B, which comprises an identical firstpartially double-stranded oligonucleotide as the above describedembodiment and a second partially double-stranded oligonucleotidecomprising: (a) a first oligonucleotide strand comprising a transposonend sequence (“ME”), a barcode sequence (“BC”), and the first primersequence (“R1”); and (b) a second strand complementary to the transposonend sequence of the second oligonucleotide strand (“ME”).

Alternatively, bead (e.g., gel bead) functionalized transposase-nucleicacid complexes are prepared as illustrated in FIG. 26 , which shows acomplex comprising a transposase, a first partially double-strandedoligonucleotide and a second double-stranded oligonucleotide. In thisembodiment, the first partially double-stranded oligonucleotidecomprises: (a) a first strand releasably attached to a bead (e.g., gelbead), wherein the first strand comprises a transposon end sequence(“ME”) and a barcode sequence (“BC”) and (b) a second strandcomplementary to the transposon end sequence of the firstoligonucleotide strand (“ME”) while the second double-strandedoligonucleotide comprises: (a) a first strand releasably attached to abead (e.g., gel bead), wherein the first strand comprises a transposonend sequence (“ME) and (b) a second strand complementary to the firstoligonucleotide strand. (“ME”). Alternative embodiments of FIG. 26comprise additional functional sequences, such as a sequencing primersequence (e.g., RI and/or R2) or an adapter sequence (e.g., P5 and/orP7).

In other embodiments, droplets are partitioned such that at least somedroplets comprise cell lysis reagents, a plurality of transposemolecules, a single cell (or nucleus), and a single bead (e.g., gelbead) comprising a barcode oligonucleotide (e.g., nucleic acid barcodemolecule) comprising a barcode sequence (“BC”) and a transposon endsequence (“ME”). See, e.g., FIGS. 25A-25B. Droplets are then subjectedto conditions such that transposase nucleic acid complexes comprising atransposase molecule and a barcode oligonucleotide are formed in thepartition.

After a transposase-nucleic acid complex is released from a bead (e.g.,gel bead) into a droplet (or is formed in the partition in embodimentssuch as FIGS. 25A-25B), the droplet is subjected to conditions such thatthe transposase-nucleic acid complexes integrate the transposon endsequences into the template nucleic acid and fragments the templatenucleic acid into double-stranded template nucleic acid fragmentsflanked by first and second partially double-stranded oligonucleotides.In alternative embodiments, cells (or nuclei) arepermeabilized/permeable and the transposase-nucleic acid complexes enterthe nuclei to fragment the template nucleic acids therein. Cells, ifpresent, are then lysed to release the double-stranded template nucleicacid fragments. Because the transposase-nucleic acid complex can onlyact on a nucleosome-free template, the double-stranded template nucleicacid fragments are representative of genome-wide areas of accessiblechromatin in a single cell or nucleus.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to fill any gaps created fromthe transposition reaction and to generate a library suitable for nextgeneration high throughput sequencing (e.g., subjecting the fragments,or derivatives thereof, to one or more reactions (e.g., nucleic acidamplification) to add functional sequences to facilitate Illuminasequencing; see, e.g., FIG. 27 ). The fully constructed library is thensequenced according to any suitable sequencing protocol.

Example 7 Generation of Barcoded Nucleic Acid Fragments from SingleCells Using Transposase-Nucleic Acid Complexes and Barcoded Adaptors(Two-Step Approach)

A plurality of transposase nucleic acid complexes and a plurality ofcells of interest (or a plurality of nuclei harvested from cells ofinterest) are partitioned such that at least some partitions (e.g.,droplets or wells) comprise a single cell (or nucleus), a plurality oftransposase nucleic acid complexes comprising a transposon end sequence,and a plurality of barcoded oligonucleotides (e.g., nucleic acid barcodemolecules) comprising a barcode sequence and a sequencing primersequence (see, e.g., FIGS. 28A-28B). In some embodiments, the pluralityof barcode oligonucleotides further comprise a transposon end sequence.In some embodiments, the plurality of barcode oligonucleotides areattached to a solid or semi-solid particle (e.g., gel bead) (see, e.g.,FIG. 28B) and partitioned such that at least some partitions (e.g.,droplets or wells) comprise a plurality of transposase nucleic acidcomplexes, a single cell (or nucleus), and a single solid or semi-solidparticle (e.g., gel bead).

For example, in some embodiments, droplet emulsion partitions aregenerated as previously described such that at least some droplets(e.g., aqueous droplets) comprise a transposase-nucleic acid complexcomprising a transposase and a pair of double-stranded oligonucleotides,cell lysis reagents, a single cell (or nucleus), and a single bead(e.g., gel bead) comprising a barcoded adaptor. Although thetransposase-nucleic acid complexes can be prepared in a variety ofdifferent configurations, a transposase-nucleic acid complex isillustrated in FIG. 28A and shows a complex comprising a transposase, afirst double-stranded oligonucleotide comprising a transposon end(“mosaic end” or “ME”) sequence, and a second double-strandedoligonucleotide comprising a transposon end (“mosaic end” or “ME”)sequence.

Cells, if present, are lysed in a manner that releases template nucleicacid molecules from into droplets (e.g., aqueous droplets), but thatsubstantially maintains native chromatin organization. Droplets are thensubjected to conditions such that the barcoded adaptors are releasedfrom beads (e.g., gel beads) into the respective droplets. Although thebarcoded adaptors can be prepared in a variety of differentconfigurations, an exemplary barcoded adaptor is illustrated in FIG. 28Band shows a single-stranded oligonucleotide comprising a transposon end(“mosaic end” or “ME”) sequence, a barcode sequence (“BC”), and a primersequence (“R1”) releasably attached to a bead (e.g., gel bead).

After barcoded adaptors are released from a bead (e.g., gel bead) into adroplet, the droplet is subjected to conditions such that thetransposase-nucleic acid complexes integrate the transposon endsequences into the template nucleic acid and fragment the templatenucleic acid into double-stranded template nucleic acid fragments. Inalternative embodiments, cells (or nuclei) are permeabilized/permeableand the transposase-nucleic acid complexes enter the nuclei to fragmentthe template nucleic acids therein. Cells, if present, are then lysed torelease the double-stranded template nucleic acid fragments. Because thetransposase-nucleic acid complex can only act on a nucleosome-freetemplate, the double-stranded template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus. After transposition and fragmentation, a PCR reactionis performed to fill any gaps created from the transposition reactionand to add the barcoded adaptors to the ends of the double-strandedtemplate nucleic acid fragments.

The double-stranded template nucleic acid fragments are then collectedfrom the droplets and processed in bulk to generate a library suitablefor next generation high throughput sequencing (e.g., subjecting thefragments, or derivatives thereof, to one or more reactions (e.g.,nucleic acid amplification) to add functional sequences to facilitateIllumina sequencing). The fully constructed library is then sequencedaccording to any suitable sequencing protocol.

Example 8 Generation of Barcoded Nucleic Acid Fragments UsingTransposase-Nucleic Acid Complexes Assembled in a Single Reaction Step

Traditional tube-based implementations of Tn5-based tagmentation systemstypically rely upon sample processing steps that take place in twoindependent reactions to generate the final transposase-fragmentednucleic acid sample. For example, in Reaction #1, oligonucleotideadaptors containing the Tn5 transposon end sequences and the Tn5transposase enzyme are incubated to form a transposase-nucleic acidcomplex. Typically, magnesium (or other divalent cations) are omittedfrom the reaction buffer to keep the transposases catalyticallyinactive. In Reaction #2, the transposase-nucleic acid complex fromReaction #1 is combined with a target double-stranded DNA and anappropriate reaction buffer containing magnesium (or other divalentcations) to activate the transposase-nucleic acid complex and causefragmentation of the target DNA and ligation of the adapteroligonucleotide sequences. While the above-described serial reactionworkflow is straightforward, implementing a tagmentation reaction withina single reaction or reaction vessel (“one-pot reaction”) can becomplicated.

A one-pot transposition reaction was performed on 50,000 HEK293T cellsand compared to a traditional two-step tagmentation reaction. Sequencinglibraries were prepared from each reaction type and sequenced on anIllumina sequencer. Sequencing data is presented in Table 1 below anddemonstrates comparable sequencing metrics between the traditional andone-pot tagmentation methods.

TABLE 1 Results from One-Pot Sequencing Reactions Sequencing metricsTraditional One-pot Total gb 43 31 Library complexity 1.1 0.52 Medianinsert size 160 180 Unmapped fraction 0.014 0.034 Non-nuclear readfraction 0.53 0.26 Fraction of fragments on target 0.57 0.81 Meanduplication rate 1.5 1.8 Percent reads gt 10 bp soft clipped 0.25 0.17

The transposition methods described herein can be utilized in a one-potreaction as described above. These one-pot reactions can be done eitherin bulk or in discrete partitions, such as a well or droplet.

Example 9 Generation of Barcoded Nucleic Acid Fragments Using BulkTagmentation and Barcoding by Ligation in Partitions (Variant 1)

Intact nuclei are harvested in bulk from cells in a cell population ofinterest in a manner that substantially maintains native chromatinorganization (e.g., using IGEPAL CA-630 mediated cell lysis). Nuclei arethen incubated in the presence of a transposase-nucleic acid complexcomprising a transposase molecule and two partially double-strandedadaptor oligonucleotides. See, e.g., FIG. 29A. Alternatively, cells arepermeable/permeabilized, allowing the transposase-nucleic acid complexto gain access to the nucleus. The first adapter oligonucleotidecomprises a double stranded transposon end sequence (“ME”) and a singlestranded Read1 sequencing primer sequence (“R1”) while the secondadapter oligonucleotide comprises a double stranded transposon endsequence (“ME”) and a single stranded Read2 sequencing primer sequence(“R2”). See FIG. 29A. In some embodiments, the R1 and/or R2 sequencingprimer in the first and/or second adapter oligonucleotide comprises aTruSeq R1 and/or R2 sequence, or a portion thereof. Thetransposase-nucleic acid complexes integrate the adaptors into thetemplate nucleic acid and produce template nucleic acid fragmentsflanked by the partially double-stranded adaptors. See FIG. 29C. Becausethe transposase-nucleic acid complex can only act on a nucleosome-freetemplate, the fragmented template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin. In someembodiments, the transposase molecules are inactivated prior to furtherprocessing steps.

Nuclei (or cells) comprising the adapter-flanked template nucleic acidfragments are then partitioned into a plurality of partitions (e.g.,droplets or wells) such that at least some partitions comprise (1) asingle nucleus (or cell) comprising the adapter-flanked template nucleicacid fragments; and (2) a plurality of partially double-stranded barcodeoligonucleotide molecules comprising a doubled stranded barcode sequence(“BC”), a double-stranded P5 adapter sequence (“P5”), and a singlestranded sequence complementary to the Read 1 sequence (“R1₁,”). SeeFIG. 29B. In some embodiments, the partially double-stranded barcodeoligonucleotide molecules are attached to a solid or semi-solid particle(e.g., a bead, such as a gel bead) and partitioned such that at leastsome partitions (e.g., droplets or wells) comprise (1) a single nucleus(or cell) and (2) a single solid or semi-solid particle (e.g., bead,such as a gel bead). In addition to the aforementioned components, insome embodiments, the plurality of partitions (e.g., droplets or wells)further comprises reagents (e.g., enzymes and buffers) that facilitatethe reactions described below.

Single cell or nucleus-containing partitions (e.g., droplets or wells)are then subjected to conditions to release adapter-flanked templatenucleic acid fragments from the nuclei (e.g., cell lysis). In certainembodiments, where barcode oligonucleotides (e.g., nucleic acid barcodemolecules) are attached to a bead (e.g., gel bead), partitions (e.g.,droplets or wells) are subjected to conditions to cause release of thebarcode oligonucleotide molecules from the bead (e.g., gel bead) (e.g.,depolymerization or degradation of the beads, for example, using areducing agent such as DTT). After release from single nuclei, theadapter-flanked template nucleic acid fragments are subjected toconditions to phosphorylate the 5′ end of the Read1 sequence (e.g.,using T4 polynucleotide kinase) for subsequent ligation steps. Afterphosphorylation, the barcode oligonucleotide molecules are ligated ontothe adapter-flanked template nucleic acid fragments using a suitable DNAligase enzyme (e.g., T4 or E. coli DNA ligase) and the complementaryRead1 sequences in the barcode oligonucleotides and the adapter-flankedtemplate nucleic acid fragments. See FIG. 29C.

After barcode ligation, gaps remaining from the transposition reactionare filled to produce barcoded, adapter-flanked template nucleic acidfragments. See FIG. 29C. The barcoded, adapter-flanked template nucleicacid fragments are then released from the partitions (e.g., droplets orwells) and processed in bulk to complete library preparation for nextgeneration high throughput sequencing (e.g., to add sample index (SI)sequences (e.g., “i7”) and/or further adapter sequences (e.g., “P7”)).In alternative embodiments, the gap filling reaction is completed inbulk after barcoded, adapter-flanked template nucleic acid fragmentshave been released from the partitions (e.g., droplets or wells). Thefully constructed library is then sequenced according to a suitablenext-generation sequencing protocol (e.g., Illumina sequencing).

Example 10 Generation of Barcoded Nucleic Acid Fragments UsingTagmentation and Barcoding by Ligation in Partitions

Cells from a cell population of interest (or nuclei from cells in a cellpopulation of interest) are partitioned into a plurality of partitions(e.g., droplets or wells) such that at least some partitions comprise(1) a single cell (or a single nucleus) comprising a template nucleicacid; and (2) a plurality of partially double-stranded barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules)comprising a doubled stranded barcode sequence (“BC”), a doubledstranded P5 adapter sequence (“P5”), and a single stranded sequencecomplementary to a Read 1 sequence (“R1,”) (e.g., FIG. 29B). In someembodiments, the partially double-stranded barcode oligonucleotidemolecules are attached to a solid or semi-solid particle (e.g., bead,such as a gel bead) and partitioned such that at least some partitions(e.g., droplets or wells) comprise (1) a single cell (or a singlenucleus) and (2) a single solid or semi-solid particle (e.g., gel bead).In addition to the aforementioned components, in some embodiments, theplurality of partitions (e.g., droplets or wells) further comprisesreagents (e.g., enzymes and buffers) that facilitate the reactionsdescribed below.

After partitioning into partitions (e.g., droplets or wells), the singlecells (or nuclei) are lysed to release the template genomic DNA in amanner that substantially maintains native chromatin organization.Partitions (e.g., droplets or wells) are then subjected to conditions togenerate a transposase-nucleic acid complex as described in Example 9and shown in FIG. 29A. Alternatively, in some embodiments, a pluralityof pre-formed transposase-nucleic acid complexes as shown in FIG. 29Aare partitioned into the plurality of partitions (e.g., droplets orwells). Partitions (e.g., droplets or wells) are then subjected toconditions such that the transposase-nucleic acid complexes integratethe first and second adapter sequences into the template nucleic acid togenerate double-stranded adapter-flanked template nucleic acidfragments. See FIG. 29D. Because the transposase-nucleic acid complexcan only act on nucleosome-free DNA, the adapter-flanked templatenucleic acid fragments are representative of genome-wide areas ofaccessible chromatin in a single cell or nucleus. Alternatively, in someembodiments, the tagmentation reaction is performed in intact nuclei,and the nuclei are lysed after transposition to release thedouble-stranded adapter-flanked template nucleic acid fragments.

Samples are then processed generally as described in Example 9. Incertain embodiments, where barcode oligonucleotides (e.g., nucleic acidbarcode molecules) are attached to a solid or semi-solid particle (e.g.,bead, such as a gel bead), droplets are subjected to conditions to causerelease of the barcode oligonucleotide molecules from the solid orsemi-solid particle (e.g., gel bead) (e.g., depolymerization ordegradation of beads, for example, using a reducing agent such as DTT).In some embodiments, the transposase molecules are inactivated (e.g., byheat inactivation) prior to further processing steps. Theadapter-flanked template nucleic acid fragments are subjected toconditions to phosphorylate the 5′ end of the Read1 sequence (e.g.,using T4 polynucleotide kinase) of the adapter-flanked template nucleicacid fragments. After phosphorylation, the barcode oligonucleotidemolecules are ligated onto the adapter-flanked template nucleic acidfragments using a suitable DNA ligase enzyme (e.g., T4, 9° N, or E. coliDNA ligase) and the complementary Read1 sequences in the barcodeoligonucleotides and the adapter-flanked template nucleic acidfragments. See FIG. 29C.

After barcode ligation, gaps remaining from the transposition reactionare filled to produce barcoded, adapter-flanked template nucleic acidfragments. See FIG. 29C. The barcoded, adapter-flanked template nucleicacid fragments are then released from the partitions (e.g., droplets orwells) and processed in bulk to complete library preparation for nextgeneration high throughput sequencing (e.g., to add sample index (SI)sequences (e.g., “i7”) and/or further adapter sequences (e.g., “P7”)).In alternative embodiments, the gap filling reaction is completed inbulk after barcoded, adapter-flanked template nucleic acid fragmentshave been released from the droplets. The fully constructed library isthen sequenced according to a suitable next-generation sequencingprotocol (e.g., Illumina sequencing).

Example 11 Generation of Barcoded Nucleic Acid Fragments Using BulkTagmentation and Barcoding by Linear Amplification in Partitions

Nuclei are harvested in bulk from cells in a cell population of interestin a manner that substantially maintains native chromatin organization.Alternatively, cells are permeabilized/permeable, allowing thetransposase-nucleic acid complex to gain access to the nucleus. Nuclei(or permeabilized cells) are then incubated in the presence of atransposase-nucleic acid complex as described in Example 9. See FIG.29A.

Nuclei (or cells) comprising the adapter-flanked template nucleic acidfragments are then partitioned into a plurality of partitions (e.g.,droplets or wells) such that at least some partitions comprise (1) asingle nucleus (or cell) comprising the adapter-flanked template nucleicacid fragments; and (2) a plurality of single-stranded barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules)comprising a transposon end sequence (“ME”), a Read1 sequence (“R1”), ora portion thereof, a barcode sequence (“BC”), and a P5 adapter sequence(“P5”). See FIG. 30A. In some embodiments, the single-stranded barcodeoligonucleotide molecules are attached to a solid or semi-solid particle(e.g., a bead, such as a gel bead) and partitioned such that at leastsome partitions (e.g., droplets or wells) comprise (1) a single nucleus(or cell) comprising the adapter-flanked template nucleic acid fragmentsand (2) a single solid or semi-solid particle (e.g., gel bead). Inaddition to the aforementioned components, in some embodiments, theplurality of partitions (e.g., droplets or wells) further comprisesreagents (e.g., enzymes and buffers) that facilitate the reactionsdescribed below.

Single cell- or nucleus-containing partitions (e.g., droplets or wells)are then subjected to conditions to release the adapter-flanked templatenucleic acid fragments from the nuclei. After the adapter-flankedtemplate nucleic acid fragments are released, gaps from thetransposition reaction are filled with a suitable enzyme. See FIG. 30B.In certain embodiments, where barcode oligonucleotides (e.g., nucleicacid barcode molecules) are attached to a solid or semi-solid particle(e.g., bead, such as a gel bead), partitions (e.g., droplets or wells)are subjected to conditions to cause release of the barcodeoligonucleotide molecules from the solid or semi-solid particle (e.g.,bead, such as a gel bead) (e.g., depolymerization or degradation ofbeads, for example, using a reducing agent such as DTT). Gap-filledadapter-flanked template nucleic acid fragments are then subjected to alinear amplification reaction using the single-stranded barcodeoligonucleotide molecules as primers to produce barcoded,adapter-flanked template nucleic acid fragments. See FIG. 30B.

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., to add sample index (SI) sequences (e.g., “i7”) and/orfurther adapter sequences (e.g., “P7”)). The fully constructed libraryis then sequenced according to a suitable next-generation sequencingprotocol (e.g., Illumina sequencing).

Example 12 Generation of Barcoded Nucleic Acid Fragments UsingTagmentation and Barcoding by Linear Amplification in Partitions

Cells from a cell population of interest (or intact nuclei from cells ina cell population of interest) are partitioned into a plurality ofpartitions (e.g., droplets or wells) such that at least some partitionscomprise (1) a single cell (or a single nucleus) comprising a templatenucleic acid; and (2) a plurality of single-stranded barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules)comprising a transposon end sequence (“ME”), a Read1 sequence (“R1”), abarcode sequence (“BC”), and a P5 adapter sequence (“P5”). See, e.g.,FIG. 30A. In some embodiments, the single-stranded barcodeoligonucleotide molecules are attached to a solid or semi-solid particle(e.g., bead, such as a gel bead) and partitioned such that at least somepartitions (e.g., droplets or wells) comprise (1) a single cell (or asingle nucleus) and (2) a single solid or semi-solid particle (e.g.,bead, such as a gel bead). In addition to the aforementioned components,in some embodiments, the plurality of partitions (e.g., droplets orwells) further comprises reagents (e.g., enzymes and buffers) thatfacilitate the reactions described below.

After partitioning into partitions (e.g., droplets or wells), the singlecells (or nuclei) are lysed to release the template genomic DNA in amanner that substantially maintains native chromatin organization. Incertain embodiments, where barcode oligonucleotides (e.g., nucleic acidbarcode molecules) are attached to a solid or semi-solid particle (e.g.,bead, such as a gel bead), partitions (e.g., droplets or wells) aresubjected to conditions to cause release of the barcode oligonucleotidemolecules from the solid or semi-solid particle (e.g., bead, such as agel bead) (e.g., depolymerization or degradation of beads, for example,using a reducing agent such as DTT). Partitions (e.g., droplets orwells) are then subjected to conditions to generate atransposase-nucleic acid complex as described in Example 9 and shown inFIG. 29A. Alternatively, in some embodiments, a plurality of pre-formedtransposase-nucleic acid complexes as shown in FIG. 29A are partitionedinto the plurality of partitions (e.g., droplets or wells). Partitions(e.g., droplets or wells) are then subjected to conditions such that thetransposase-nucleic acid complexes integrate the first and secondadapter sequences into the template nucleic acid to generatedouble-stranded adapter-flanked template nucleic acid fragments. Becausethe transposase-nucleic acid complex can only act on nucleosome-freeDNA, the adapter-flanked template nucleic acid fragments arerepresentative of genome-wide areas of accessible chromatin in a singlecell or nucleus. Alternatively, in some embodiments, the tagmentationreaction is performed in intact nuclei, and the nuclei are lysed torelease the double-stranded adapter-flanked template nucleic acidfragments.

Samples are then processed generally as described in Example 11. Aftertagmentation, gaps from the transposition reaction are filled with asuitable gap-filling enzyme. See FIG. 31 . Gap-filled adapter-flankedtemplate nucleic acid fragments are then subjected to a linearamplification reaction using the single-stranded barcode oligonucleotidemolecules as primers to produce barcoded, adapter-flanked templatenucleic acid fragments. See FIG. 31 .

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., to add sample index (SI) sequences (e.g., “i7”) and/orfurther adapter sequences (e.g., “P7”)). The fully constructed libraryis then sequenced according to a suitable next-generation sequencingprotocol (e.g., Illumina sequencing).

Example 13 Generation of Barcoded Nucleic Acid Fragments using BulkTagmentation and CRISPR/Cas9 Cleavage in Partitions

Nuclei are harvested in bulk from cells in a cell population of interestin a manner that substantially maintains native chromatin organization.Alternatively, cells are permeabilized/permeable, allowing thetransposase-nucleic acid complex to gain access to the nucleus. Nucleiare then incubated in the presence of a transposase-nucleic acid complexas described in Example 9. See FIG. 29A. In some embodiments, aftertransposition, the transposase is inactivated or dissociated from theadapter-flanked template nucleic acid fragments.

Nuclei (or cells) comprising the adapter-flanked template nucleic acidfragments are then partitioned into a plurality of partitions (e.g.,droplets or wells) such that at least some partitions comprise (1) asingle nucleus comprising the adapter-flanked template nucleic acidfragments; (2) a plurality of double-stranded barcode oligonucleotidemolecules (e.g., nucleic acid barcode molecules) comprising a barcodesequence (BC) and a TruSeqR1 sequencing primer sequence (e.g., FIG.32A); and (3) a plurality of CRISPR/Cas9 complexes comprising a Cas9nuclease and a synthetic guide RNA (gRNA) that targets the Read1/MEadapter sequence in the adapter-flanked template nucleic acid fragments.See FIG. 32B. In some embodiments, the double-stranded barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules) areattached to a solid or semi-solid particle (e.g., bead, such as a gelbead) and partitioned such that at least some droplets comprise (1) asingle nucleus; (2) a single solid or semi-solid particle (e.g., gelbead); and (3) a plurality of CRISPR/Cas9 complexes. In addition to theaforementioned components, in some embodiments, the plurality ofpartitions (e.g., droplets or wells) further comprises reagents (e.g.,enzymes and buffers) that facilitate the reactions described below.

Single nucleus-containing partitions (e.g., droplets or wells) are thensubjected to conditions to release the adapter-flanked template nucleicacid fragments from the nuclei. After the adapter-flanked templatenucleic acid fragments are released, gaps from the transpositionreaction are filled with a suitable gap-filling enzyme. Gap-filledadapter-flanked template nucleic acid fragments are then subjected toCas9-mediated cleavage of the R1/ME adaptor, or some portion thereof.See FIG. 32B. In certain embodiments, where barcode oligonucleotides(e.g., nucleic acid barcode molecules) are attached to a solid orsemi-solid particle (e.g., a bead, such as a gel bead), partitions(e.g., droplets or wells) are subjected to conditions to cause releaseof the barcode oligonucleotide molecules from the solid or semi-solidparticle (e.g., gel bead) (e.g., depolymerization or degradation ofbeads, for example, using a reducing agent such as DTT). The barcodeoligonucleotides are then ligated onto the R1 adapter-cleaved ends ofthe template nucleic acid fragments to produce barcoded, adapter-flankedtemplate nucleic acid fragments. See FIG. 32B.

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., subjecting the fragments, or derivatives thereof, toone or more reactions (e.g., nucleic acid amplification) to addfunctional sequences to facilitate Illumina sequencing). In someembodiments, a second CRISPR/Cas9 mediated cleavage event using asynthetic guide RNA (gRNA) that targets the Read2/ME adapter sequence isperformed either in the partitions or in bulk after release from thepartitions. The fully constructed library is then sequenced according toany suitable sequencing protocol.

Example 14 Generation of Barcoded Nucleic Acid Fragments UsingTagmentation and CRISPR/Cas9 Cleavage in Partitions

Cells from a cell population of interest (or intact nuclei from cells ina cell population of interest) are partitioned into a plurality ofpartitions (e.g., droplets or wells) such that at least some partitionscomprise (1) a single cell (or a single nucleus) comprising a templatenucleic acid; (2) a plurality of double-stranded barcode oligonucleotidemolecules (e.g., nucleic acid barcode molecules) comprising a barcodesequence (“BC”) and a TruSeqR1 sequencing primer sequence (e.g., FIG.32A); and (3) a plurality of CRISPR/Cas9 complexes comprising a Cas9nuclease and a synthetic guide RNA (gRNA) that targets the Read1/MEadapter sequence in the adapter-flanked template nucleic acid fragments.See, e.g., FIG. 32C. In some embodiments, the double-stranded barcodeoligonucleotide molecules (e.g., nucleic acid barcode molecules) areattached to a solid or semi-solid particle (e.g., bead, such as a gelbead) and partitioned such that at least some partitions (e.g., dropletsor wells) comprise (1) a single cell (or single nucleus); (2) a singlesolid or semi-solid particles (e.g., gel bead); and (3) a plurality ofCRISPR/Cas9 complexes. In addition to the aforementioned components, insome embodiments, the plurality of partitions (e.g., droplets or wells)further comprises reagents (e.g., enzymes and buffers) that facilitatethe reactions described below.

After partitioning into partitions (e.g., droplets or wells), the singlecells (or nuclei) are lysed to release the template genomic DNA in amanner that substantially maintains native chromatin organization.Partitions (e.g., droplets or wells) are then subjected to conditions togenerate a transposase-nucleic acid complex as described in Example 9and shown in FIG. 29A. Alternatively, in some embodiments, a pluralityof pre-formed transposase-nucleic acid complexes as shown in FIG. 29Aare partitioned into the plurality of partitions (e.g., droplets orwells). Partitions (e.g., droplets or wells) are then subjected toconditions such that the transposase-nucleic acid complexes integratethe first and second adapter sequences into the template nucleic acid togenerate double-stranded adapter-flanked template nucleic acidfragments. Because the transposase-nucleic acid complex can only act onnucleosome-free DNA, the adapter-flanked template nucleic acid fragmentsare representative of genome-wide areas of accessible chromatin in asingle cell or nucleus. Alternatively, in some embodiments, thetagmentation reaction is performed in intact nuclei, and the nuclei arelysed to release the double-stranded adapter-flanked template nucleicacid fragments.

Samples are then processed generally as described in Example 13. Aftertagmentation, gaps from the transposition reaction are filled with asuitable gap-filling enzyme. See FIG. 32C. Gap-filled adapter-flankedtemplate nucleic acid fragments are then subjected to Cas9-mediatedcleavage of the R1 adaptor. See FIG. 32C. In certain embodiments, wherebarcode oligonucleotides (e.g., nucleic acid barcode molecules) areattached to a solid or semi-solid particle (e.g., bead, such as a gelbead), partitions (e.g., droplets or wells) are subjected to conditionsto cause release of the barcode oligonucleotide molecules from the solidor semi-solid particle (e.g., gel bead) (e.g., depolymerization ordegradation of beads, for example, using a reducing agent such as DTT).The barcode oligonucleotides are then ligated onto the R1adapter-cleaved ends of the template nucleic acid fragments to producebarcoded, adapter-flanked template nucleic acid fragments. See FIG. 32C.

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., subjecting the fragments, or derivatives thereof, toone or more reactions (e.g., nucleic acid amplification) to addfunctional sequences to facilitate Illumina sequencing). In someembodiments, a second CRISPR/Cas9 mediated cleavage event using asynthetic guide RNA (gRNA) that targets the Read2/ME adapter sequence isperformed either in the partitions or in bulk after release from thepartitions. The fully constructed library is then sequenced according toany suitable sequencing protocol.

Example 15 Generation of Barcoded Nucleic Acid Fragments Using BulkTagmentation and CRISPR/Cas9 Cleavage in Partitions Using Y-Adapters

Nuclei are harvested in bulk from cells in a cell population of interestin a manner that substantially maintains native chromatin organization.Alternatively, cells are permeabilized/permable, allowing thetransposase-nucleic acid complex to gain access to the nucleus. Nucleiare then incubated in the presence of a transposase-nucleic acid complexas described in Example 9. See FIG. 29A. In some embodiments, aftertransposition, the transposase is inactivated or dissociated from theadapter-flanked template nucleic acid fragments.

Nuclei (or cell) comprising the adapter-flanked template nucleic acidfragments are then partitioned into a plurality of partitions (e.g.,droplets or wells) such that at least some partitions comprise (1) asingle nucleus comprising the adapter-flanked template nucleic acidfragments; (2) a plurality of Y-adaptor barcode oligonucleotidemolecules (e.g., nucleic acid barcode molecules) comprising a barcodesequence (“BC”), a Read1 sequencing primer sequence (“R1”), and a Read2sequencing primer sequence (“R2”), e.g., FIG. 33A; (3) a first pluralityof CRISPR/Cas9 complexes comprising a Cas9 nuclease and a syntheticguide RNA (gRNA) that targets the Read1/ME adapter sequence in theadapter-flanked template nucleic acid fragments; and (4) a secondplurality of CRISPR/Cas9 complexes comprising a Cas9 nuclease and asynthetic guide RNA (gRNA) that targets the Read2/ME adapter sequence inthe adapter-flanked template nucleic acid fragments. See FIG. 33C. Insome embodiments, the Y-adaptor barcode oligonucleotide molecules areattached to a solid or semi-solid particle (e.g., a bead, such as a gelbead) (e.g., FIG. 15A) and partitioned such that at least somepartitions (e.g., droplets or wells) comprise (1) a single nucleus; (2)a single solid or semi-solid particle (e.g., bead, such as a gel bead);(3) the first plurality of CRISPR/Cas9 complexes; and (4) the secondplurality of CRISPR/Cas9 complexes. In addition to the aforementionedcomponents, in some embodiments, the plurality of partitions (e.g.,droplets or wells) further comprises reagents (e.g., enzymes andbuffers) that facilitate the reactions described below.

Single nucleus containing partitions (e.g., droplets or wells) are thensubjected to conditions to release the adapter-flanked template nucleicacid fragments from the nuclei. After the adapter-flanked templatenucleic acid fragments are released, gaps from the transpositionreaction are filled with a suitable gap-filling enzyme. Gap-filledadapter-flanked template nucleic acid fragments are then subjected toCas9-mediated cleavage of the R1 and R2 adaptors, or a portion thereof.See FIG. 33B. In certain embodiments, where barcode oligonucleotides(e.g., nucleic acid barcode molecules) are attached to a solid orsemi-solid particle (e.g., a bead, such as a gel bead), partitions(e.g., droplets or wells) are subjected to conditions to cause releaseof the barcode oligonucleotide molecules from the solid or semi-solidparticle (e.g., gel bead) (e.g., depolymerization or degradation ofbeads, for example, using a reducing agent such as DTT). The Y-adaptorbarcode oligonucleotides are then ligated onto the R1/R2 adapter-cleavedends of the template nucleic acid fragments to produce barcoded,adapter-flanked template nucleic acid fragments. See FIG. 33B.

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., subjecting the fragments, or derivatives thereof, toone or more reactions (e.g., nucleic acid amplification) to addfunctional sequences to facilitate Illumina sequencing). The fullyconstructed library is then sequenced according to any suitablesequencing protocol.

Example 16 Generation of Barcoded Nucleic Acid Fragments UsingTagmentation and CRISPR/Cas9 Cleavage in Partitions Using Y-Adapters

Cells from a cell population of interest (or intact nuclei from cells ina cell population of interest) are partitioned into a plurality ofpartitions (e.g., droplets or wells) such that at least some partitionscomprise (1) a single cell (or a single nucleus) comprising a templatenucleic acid; (2) a plurality of Y-adaptor barcode oligonucleotidemolecules (e.g., nucleic acid barcode molecules) comprising a barcodesequence (“BC”), a Read1 sequencing primer sequence (“R1”), and a Read2sequencing primer sequence (“R2”) (e.g., FIG. 33A); (3) a firstplurality of CRISPR/Cas9 complexes comprising a Cas9 nuclease and asynthetic guide RNA (gRNA) that targets the Read1/ME adapter sequence inthe adapter-flanked template nucleic acid fragments; and (4) a secondplurality of CRISPR/Cas9 complexes comprising a Cas9 nuclease and asynthetic guide RNA (gRNA) that targets the Read2/ME adapter sequence inthe adapter-flanked template nucleic acid fragments. See FIG. 33C. Insome embodiments, the Y-adaptor barcode oligonucleotide molecules areattached to a solid or semi-solid particle (e.g., a bead, such as a gelbead) and partitioned such that at least some partitions (e.g., dropletsor wells) comprise (1) a single cell (or single nucleus); (2) a singlesolid or semi-solid particle (e.g., gel bead); (3) the first pluralityof CRISPR/Cas9 complexes; and (4) the second plurality of CRISPR/Cas9complexes. In addition to the aforementioned components, in someembodiments, the plurality of partitions (e.g., droplets or wells)further comprises reagents (e.g., enzymes and buffers) that facilitatethe reactions described below.

After partitioning into partitions (e.g., droplets or wells), the singlecells (or nuclei) are lysed to release the template genomic DNA in amanner that substantially maintains native chromatin organization.Partitions (e.g., droplets or wells) are then subjected to conditions togenerate a transposase-nucleic acid complex as described in Example 9and shown in FIG. 29A. Alternatively, in some embodiments, a pluralityof pre-formed transposase-nucleic acid complexes as shown in FIG. 29Aare partitioned into the plurality of partitions (e.g., droplets orwells). Partitions (e.g., droplets or wells) are then subjected toconditions such that the transposase-nucleic acid complexes integratethe first and second adapter sequences into the template nucleic acid togenerate double-stranded adapter-flanked template nucleic acidfragments. Because the transposase-nucleic acid complex can only act onnucleosome-free DNA, the adapter-flanked template nucleic acid fragmentsare representative of genome-wide areas of accessible chromatin in asingle cell or nucleus. Alternatively, in some embodiments, thetagmentation reaction is performed in intact nuclei, and the nuclei arelysed to release the double-stranded adapter-flanked template nucleicacid fragments.

Samples are then processed generally as described in Example 15. Aftertagmentation, gaps from the transposition reaction are filled with asuitable gap-filling enzyme. See FIG. 33C. Gap-filled adapter-flankedtemplate nucleic acid fragments are then subjected to Cas9-mediatedcleavage of the R1 and R2 adaptors, or a portion thereof. See FIG. 33C.In certain embodiments, where barcode oligonucleotides (e.g., nucleicacid barcode molecules) are attached to a solid or semi-solid particle(e.g., a bead, such as a gel bead), partitions (e.g., droplets or wells)are subjected to conditions to cause release of the barcodeoligonucleotide molecules from the solid or semi-solid particle (e.g.,gel bead) (e.g., depolymerization or degradation of beads, for example,using a reducing agent such as DTT). The Y-adaptor barcodeoligonucleotides are then ligated onto the R1/R2 adapter-cleaved ends ofthe template nucleic acid fragments to produce barcoded, adapter-flankedtemplate nucleic acid fragments. See FIG. 33C.

The barcoded, adapter-flanked template nucleic acid fragments are thenreleased from the partitions (e.g., droplets or wells) and processed inbulk to complete library preparation for next generation high throughputsequencing (e.g., subjecting the fragments, or derivatives thereof, toone or more reactions (e.g., nucleic acid amplification) to addfunctional sequences to facilitate Illumina sequencing). The fullyconstructed library is then sequenced according to any suitablesequencing protocol.

Example 17 Generation of Barcoded Nucleic Acid Molecules for ATAC-seqand Barcoded cDNA from the Same Single Cell

Cells from a cell population of interest (or intact nuclei from cells ina cell population of interest) are partitioned into a plurality ofpartitions (e.g., droplets or wells) such that at least some partitionscomprise (1) a single cell (or a single nucleus) comprising templategenomic DNA molecules and template RNA molecules; (2) a plurality offirst barcoded oligonucleotide molecules (e.g., first nucleic acidbarcode molecules) comprising a barcode sequence; (3) a plurality oftransposase molecules, (4) a plurality of second barcode oligonucleotidemolecules (e.g., second nucleic acid barcode molecules) comprising abarcode sequence and a capture sequence; and (5) a plurality of reversetranscriptase molecules. In some embodiments, the barcode sequence fromthe first barcoded oligonucleotide and the barcode sequence from thesecond barcoded oligonucleotide is the same. In some embodiments, thebarcode sequence from the first barcoded oligonucleotide and the barcodesequence from the second barcoded oligonucleotide are different.

In some embodiments, the plurality of first barcoded oligonucleotides(e.g., first nucleic acid barcode molecules) and the plurality of secondbarcoded oligonucleotides (e.g., second nucleic acid barcode molecules)are attached to a solid or semi-solid particle (e.g., a bead, such as agel bead) and partitioned such that at least some partitions (e.g.,droplets or wells) comprise (1) a single cell (or single nucleus); (2) asingle solid or semi-solid particle (e.g., gel bead) comprising thefirst and second plurality of barcoded oligonucleotides; (3) a pluralityof transposase molecules; and (4) a plurality of reverse transcriptasemolecules. In other embodiments, the plurality of first barcodedoligonucleotides (e.g., first nucleic acid barcode molecules) areattached to a first solid or semi-solid particle (e.g., bead, such as agel bead) while the plurality of second barcoded oligonucleotides (e.g.,second nucleic acid barcode molecules) are attached to a second solid orsemi-solid particle (e.g., bead, such as a gel bead) and partitionedsuch that at least some partitions (e.g., droplets or wells) comprise(1) a single cell (or single nucleus); (2) a single first solid orsemi-solid particle (e.g., gel bead); (2) a single second solid orsemi-solid particle (e.g., gel bead); (3) a plurality of transposasemolecules; and (4) a plurality of reverse transcriptase molecules.

In certain embodiments, the plurality of first barcoded oligonucleotides(e.g., first nucleic acid barcode molecules) are attached to a pluralityof solid or semi-solid particles (e.g., beads, such as gel beads) whilethe plurality of second barcoded oligonucleotides (e.g., second nucleicacid barcode molecules) are attached to a plurality of magneticparticles (e.g., beads), wherein the plurality of magnetic particles areembedded within the solid or semi-solid particles (e.g., gel beads).Continuing these embodiments, the plurality of solid or semi-solidparticles (e.g., gel beads) is partitioned such that at least somepartitions (e.g., droplets or wells) comprise: (1) a single cell (orsingle nucleus); (2) a single solid or semi-solid particle (e.g., gelbead) comprising (i) a plurality of first barcoded oligonucleotidesattached to the single solid or semisolid particle (e.g., gel bead); and(ii) a plurality of magnetic particles embedded within the single solidor semi-solid particle (e.g., gel bead), wherein the magnetic particlescomprise the second barcode oligonucleotide attached thereto; (3) aplurality of transposase molecules; and (4) a plurality of reversetranscriptase molecules. See, e.g., FIG. 34 . Similarly, in otherembodiments, the second barcode oligonucleotides (e.g., second nucleicacid barcode molecules) are attached to the solid or semi-solid particle(e.g., gel bead) while the first oligonucleotides (e.g., first nucleicacid barcode molecules) are attached to a plurality of magneticparticles embedded within the solid or semi-solid particle (e.g., gelbead). See, e.g., FIG. 34 . In addition to the aforementionedcomponents, in some embodiments, the plurality of partitions (e.g.,droplets or wells) further comprise reagents (e.g., enzymes and buffers)that facilitate the reactions described herein.

The first barcoded oligonucleotide and related nucleic acid processingsteps can take on the structure of any of the aforementioned Examplesand may include additional components as described therein. Forinstance, in some embodiments, the first barcoded oligonucleotidecomprises a barcode sequence and a transposon end sequence (e.g., a MEsequence) and is, for example, (1) a forked adapter such as thosedescribed in Example 1, FIGS. 12A-12B; (2) a T7-containingoligonucleotide such as those described in Example 3, FIG. 18 ; or (3) abarcoded oligonucleotide such as those described in (i) Example 7, FIG.28B; (ii) Example 11, FIGS. 30A-30B; and (iii) Example 12, FIG. 31 . Inother embodiments, the first barcoded oligonucleotide comprises abarcode sequence and is, for example, (1) a forked adapter such as thosedescribed in Example 2, FIGS. 15A-15B or Examples 15-16, FIGS. 33A-33C;or (2) a barcoded oligonucleotide such as those described in Examples 9and 10, FIGS. 29A-29C or Examples 13-14, FIGS. 32A-32C. In some cases,the second barcoded oligonucleotide comprises a second barcode sequenceand a capture sequence, where the capture sequence can be, for example,a poly-T sequence, a random primer sequence (e.g., a random hexamer), ora gene-specific sequence.

After partitioning into partitions (e.g., droplets or wells), the singlecells (or nuclei) are lysed to release template genomic DNA and templateRNA (e.g., cytoplasmic mRNA or nuclear mRNA) in a manner thatsubstantially maintains native chromatin organization of the genomicDNA. In certain embodiments, where barcode oligonucleotides are attachedto a solid or semi-solid particle (e.g., a bead, such as a gel bead),partitions (e.g., droplets or wells) are subjected to conditions tocause release of barcode oligonucleotide molecules from the solid orsemi-solid particle (e.g., gel bead) (e.g., depolymerizati on ordegradation of beads, for example, using a reducing agent such as DTT).Partitions (e.g., droplets or wells) are then subjected to conditions togenerate a transposase-nucleic acid complex as described in theaforementioned examples. Alternatively, in some embodiments, a pluralityof pre-formed transposase-nucleic acid complexes are partitioned intothe plurality of (e.g., droplets or wells). Partitions (e.g., dropletsor wells) are then subjected to conditions such that thetransposase-nucleic acid complexes generate double-stranded templategenomic DNA fragments. As described above, the transposition reactioncan take on the structure of any of the aforementioned Examples togenerate double-stranded template genomic DNA fragments flanked by awide variety of functional sequences and suitable for a number ofdownstream processing steps. For example, as described herein, in someembodiments, the transposition reaction directly integrates the barcodesequence into the template genomic DNA fragments (e.g., Example 1)while, in other embodiments, the barcode sequence is added to templategenomic DNA fragments subsequent to the transposition reaction (such asby ligation, e.g., Example 2). Because the transposase-nucleic acidcomplex can only act on nucleosome-free DNA, the template genomic DNAfragments are representative of genome-wide areas of accessiblechromatin in a single cell or nucleus. Alternatively, in someembodiments, the tranposition reaction is performed in intact nuclei,and the nuclei are lysed to release the adapter-flanked template genomicDNA fragments. Alternatively, in some embodiments, the transpositionreaction is performed in bulk in intact nuclei and a single nucleuscomprising template genomic DNA fragments is partitioned and processedas described above. In some embodiments, gaps from the transpositionreaction are filled in-droplet with a suitable gap-filling enzyme. Inother embodiments, a gap-filling reaction is performed in bulk after thedouble-stranded, barcoded adapter-flanked DNA fragments have beenreleased from the partitions.

In some cases, partitions (e.g., droplets or wells) may be subjected toconditions to capture template RNA (e.g., messenger RNA, mRNA) on secondsolid or semi-solid particles using a capture sequence of secondbarcoded oligonucleotides of the plurality of second barcodedoligonucleotides. Alternatively, second barcoded oligonucleotides of theplurality of second barcoded oligonucleotides may be released from thesecond solid or semi-solid particles (e.g., as described herein) andused to capture template RNA. Captured template RNA may be subsequentlyprocessed in bulk after removal from the partitions. Alternatively, insome cases, partitions (e.g., droplets or wells) may be subjected toconditions to generate single-stranded, barcoded cDNA molecules from thetemplate RNA using the capture sequence from the second barcodeoligonucleotide to prime the reverse transcription reaction (e.g., anoligo (dT) sequence). In some embodiments, second strand cDNA isproduced (e.g., through a template switching oligonucleotide or throughrandom priming) to generate double-stranded, barcoded cDNA molecules. Insome embodiments, the template switching oligonucleotide also comprisesa barcode sequence such that both the 5′ and 3′ end of the cDNA comprisea barcode sequence. The barcode sequence on the 5′ and 3′ end can be thesame barcode sequence or the 5′ end can have a different barcodesequence than the 3′ end. In other embodiments, the plurality of secondbarcode oligonucleotide molecules are omitted and replaced withplurality of second oligonucleotide molecules comprising a capturesequence and no barcode sequence. Continuing with these embodiments,first strand cDNA molecules are generated using the capture sequencewhile second strand cDNA is generated through use of a barcoded templateswitching oligonucleotide to barcode the 5′ end of the template RNA. Insome embodiments, an in-partition (e.g., in-droplet or in-well)amplification reaction, such as linear amplification, is performed onthe adapter-flanked DNA fragments, the barcoded cDNA molecules, or boththe adapter-flanked DNA fragments and the barcoded cDNA molecules. Insome embodiments, a barcode oligonucleotide is directly ligated onto thetemplate RNA.

Exemplary Scheme 1—scATAC-seq+3′ scRNA-seq using ligation

In an exemplary barcoding scheme, cells from a cell population ofinterest (or intact nuclei from cells in a cell population of interest)are partitioned into a plurality of partitions (e.g., droplets or wells)such that at least some partitions comprise: (1) a single cell (or asingle nucleus) comprising template genomic DNA molecules and templateRNA molecules (e.g., mRNA or nuclear pre-mRNA); (2) a solid orsemi-solid particle (e.g., bead, such as a gel bead) comprising (i) aplurality of partially double-stranded nucleic acid barcode molecules asshown in FIG. 29B (see also FIG. 35A), and (ii) a plurality of nucleicacid barcode molecules as shown in FIG. 35B; (3) a plurality oftransposase molecules; (4) a plurality of nucleic acid moleculescomprising a transposase end sequence (e.g., FIG. 29A); and (5) aplurality of reverse transcriptase molecules. In some embodiments, thesolid or semi-solid particle is a bead. In some embodiments, the bead isa gel bead. In some embodiments, the solid or semi-solid particle (e.g.,gel bead) comprises the plurality of partially double-stranded nucleicacid barcode molecules of FIG. 35A attached thereto and a plurality ofmagnetic particles embedded within the solid or semi-solid particle(e.g., gel bead) (see, e.g., FIG. 34 ), wherein the plurality ofmagnetic particles comprises, attached thereto, the plurality of nucleicacid barcode molecules of FIG. 35B. In other embodiments, the solid orsemi-solid particle (e.g., gel bead) comprises the plurality of nucleicacid barcode molecules of FIG. 35B attached thereto and a plurality ofmagnetic particles embedded within the solid or semi-solid particle(e.g., gel bead), wherein the plurality of magnetic particles comprises,attached thereto, the plurality of nucleic acid barcode molecules ofFIG. 35A. In some embodiments, the barcode sequence in FIG. 35A and thebarcode sequence FIG. 35B is the same. In some embodiments, the barcodesequence in FIG. 35A and the barcode sequence FIG. 35B comprise one ormore barcode segments that are identical. In some embodiments, thebarcode sequence in FIG. 35A and the barcode sequence FIG. 35B aredifferent.

Continuing these embodiments, after partitioning, the single cells (ornuclei) are lysed to release template genomic DNA and template RNA(e.g., cytoplasmic mRNA or nuclear mRNA) in a manner that substantiallymaintains native chromatin organization of the genomic DNA. In certainembodiments, the plurality of nucleic acid barcode molecules of FIG. 35Aand FIG. 35B are releasably attached to the solid or semi-solid particle(e.g., bead) and partitions (e.g., droplets or wells) are subjected toconditions to cause release of barcode oligonucleotide molecules fromthe solid or semi-solid particle (e.g., bead) (e.g., depolymerization ordegradation of a bead, for example, using a reducing agent such as DTT).Partitions (e.g., droplets or wells) are then subjected to conditions togenerate a transposase-nucleic acid complex as described in theaforementioned examples. Alternatively, in some embodiments, a pluralityof pre-formed transposase-nucleic acid complexes (e.g., FIG. 29A) arepartitioned into the plurality of partitions (e.g., droplets or wells).Partitions are then subjected to conditions such that thetransposase-nucleic acid complexes generate double-stranded templategenomic DNA fragments. Alternatively, in some embodiments, thetransposition reaction is performed in bulk in intact nuclei which arepartitioned such that at least some partitions (e.g., droplets or wells)comprise (1) a single cell (or a single nucleus) comprising templategenomic DNA fragments and template RNA molecules; (2) a solid orsemi-solid particle (e.g., a bead, such as a gel bead) comprising (i) aplurality of partially double-stranded nucleic acid barcode molecules asshown in FIG. 29B (see also FIG. 35A), and (ii) a plurality of nucleicacid barcode molecules as shown in FIG. 35B; and (3) a plurality ofreverse transcriptase molecules. In some embodiments, the nucleic acidbarcode molecules of FIG. 35A or FIG. 35B are attached to a magneticparticle embedded within a solid or semi-solid particle (e.g., a bead,such as a gel bead), as previously described. After partitioning, thesingle nuclei are lysed to release template genomic DNA fragments andtemplate RNA (e.g., cytoplasmic mRNA or nuclear mRNA) molecules.Continuing this embodiment, after fragmentation, template genomic DNAfragments are processed generally as outlined in FIG. 29D (or FIG. 29Cfor bulk tagmentation) by ligating a partially double-stranded nucleicacid barcode molecule as shown in FIG. 29B (see also FIG. 35A) to thetemplate nucleic acid fragments (see, e.g., Examples 9 and 10). Usingthe reverse transcriptase, template RNA molecules comprising a poly-Atail (e.g., mRNA molecules) are processed with the plurality of nucleicacid barcode molecules of FIG. 35B to generate barcoded cDNA moleculesas generally described elsewhere herein.

Exemplary Scheme 2—scATAC-seq−H 5′ scRATA-seq using ligation

In another barcoding scheme, cells from a cell population of interest(or intact nuclei from cells in a cell population of interest) arepartitioned into a plurality of partitions (e.g., droplets or wells)such that at least some partitions comprise: (1) a single cell (or asingle nucleus) comprising template genomic DNA molecules and templateRNA molecules (e.g., mRNA or nuclear pre-mRNA); (2) a solid orsemi-solid particle (e.g., bead, such as a gel bead) comprising (i) aplurality of partially double-stranded nucleic acid barcode molecules asshown in FIG. 29B (see also FIG. 35A), and (ii) a plurality of nucleicacid barcode molecules as shown in FIG. 35D; (3) a plurality oftransposase molecules; (4) a plurality of nucleic acid moleculescomprising a transposase end sequence (e.g., FIG. 29A); (5) a pluralityof nucleic acid molecules comprising a poly-T sequence; and (6) aplurality of reverse transcriptase molecules. In some embodiments, thesolid or semi-solid particle is a bead. In some embodiments, the bead isa gel bead. In some embodiments, the solid or semi-solid particle (e.g.,gel bead) comprises the plurality of partially double-stranded nucleicacid barcode molecules of FIG. 35A attached thereto and a plurality ofmagnetic particles embedded within the solid or semi-solid particle(e.g., gel bead) (see, e.g., FIG. 34 ), wherein the plurality ofmagnetic particles comprises, attached thereto, the plurality of nucleicacid barcode molecules of FIG. 35D. In other embodiments, the solid orsemi-solid particle (e.g., gel bead) comprises the plurality of nucleicacid barcode molecules of FIG. 35D attached thereto and a plurality ofmagnetic particles embedded within the solid or semi-solid particle(e.g., gel bead), wherein the plurality of magnetic particles comprises,attached thereto, the plurality of nucleic acid barcode molecules ofFIG. 35A. In some embodiments, the barcode sequence in FIG. 35A and thebarcode sequence FIG. 35D is the same. In some embodiments, the barcodesequence in FIG. 35A and the barcode sequence FIG. 35D comprise one ormore barcode segments that are identical. In some embodiments, thebarcode sequence in FIG. 35A and the barcode sequence FIG. 35D aredifferent.

Continuing these embodiments, after partitioning, the single cells (ornuclei) are lysed to release template genomic DNA and template RNA(e.g., cytoplasmic mRNA or nuclear mRNA) in a manner that substantiallymaintains native chromatin organization of the genomic DNA. In certainembodiments, the plurality of nucleic acid barcode molecules of FIG. 35Aand FIG. 35D are releasably attached to the solid or semi-solid particle(e.g., bead, such as a gel bead) and partitions (e.g., droplets orwells) are subjected to conditions to cause release of barcodeoligonucleotide molecules from the bead (e.g., depolymerization ordegradation of a bead, for example, using a reducing agent such as DTT).Partitions are then subjected to conditions to generate atransposase-nucleic acid complex as described in the aforementionedexamples. Alternatively, in some embodiments, a plurality of pre-formedtransposase-nucleic acid complexes (e.g., FIG. 29A) are partitioned intothe plurality of partitions (e.g., droplets or wells). Partitions arethen subjected to conditions such that the transposase-nucleic acidcomplexes generate double-stranded template genomic DNA fragments.Alternatively, in some embodiments, the transposition reaction isperformed in bulk in intact nuclei which are partitioned such that atleast some partitions comprise (1) a single cell (or a single nucleus)comprising template genomic DNA fragments and template RNA molecules;(2) a solid or semi-solid particle (e.g., a bead, such as a gel bead)comprising (i) a plurality of partially double-stranded nucleic acidbarcode molecules as shown in FIG. 29B (see also FIG. 35A), and (ii) aplurality of nucleic acid barcode molecules as shown in FIG. 35D; (3) aplurality of nucleic acid molecules comprising a poly-T sequence; and(4) a plurality of reverse transcriptase molecules. In some embodiments,the nucleic acid barcode molecules of FIG. 35A or FIG. 35D are attachedto a magnetic particle embedded within a solid or semi-solid particle(e.g., gel bead), as previously described. After partitioning, thesingle nuclei are lysed to release template genomic DNA fragments andtemplate RNA (e.g., cytoplasmic mRNA or nuclear mRNA) molecules.Continuing this embodiment, after fragmentation, template genomic DNAfragments are processed generally as outlined in FIG. 29D (or FIG. 29Cfor bulk tagmentation) by ligating a partially double-stranded nucleicacid barcode molecule as shown in FIG. 29B (see also FIG. 35A) to thetemplate nucleic acid fragments (see, e.g., Examples 9 and 10). TemplateRNA molecules (e.g., mRNA molecules) are processed to generate 5′barcoded cDNA molecules. For example, a template RNA molecule comprisinga poly-A tail (e.g., mRNA) and a nucleic acid molecule comprising apoly-T sequence are processed using the reverse transcriptase togenerate a cDNA molecule. In an example of template switching, in someembodiments, an enzyme with terminal transferase activity (e.g., areverse transcriptase with terminal transferase activity) addsadditional nucleotides, (e.g., polyC), to the cDNA in a templateindependent manner. Switch oligonucleotides or switch oligonucleotidesequences can include sequences complementary to the additionalnucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) onthe cDNA hybridize to the additional nucleotides (e.g., polyG) on theswitch oligo, whereby the switch oligo can be used by the reversetranscriptase as template to further extend the cDNA. Thus, in someembodiments, the reverse transcriptase comprises terminal transferaseactivity and generates 5′ barcoded cDNA fragments using the switcholigonucleotide sequence in the plurality of nucleic acid barcodemolecules of FIG. 35D.

Exemplary Scheme 3 scATAC-seq 3′ scR1VA-seq using linear amplification

In certain barcoding schemes, cells from a cell population of interest(or intact nuclei from cells in a cell population of interest) arepartitioned into a plurality of partitions (e.g., droplets) such that atleast some partitions comprise: (1) a single cell (or a single nucleus)comprising template genomic DNA molecules and template RNA molecules(e.g., mRNA or nuclear pre-mRNA); (2) a solid or semi-solid particle(e.g., a bead, such as a gel bead) comprising (i) a plurality ofsingle-stranded nucleic acid barcode molecules as shown in FIG. 35C, and(ii) a plurality of nucleic acid barcode molecules as shown in FIG. 35B;(3) a plurality of transposase molecules; (4) a plurality of nucleicacid molecules comprising a transposase end sequence (e.g., FIG. 29A);and (5) a plurality of reverse transcriptase molecules. In someembodiments, the solid or semi-solid particle is a bead. In someembodiments, the bead is a gel bead. In some embodiments, the solid orsemi-solid particle (e.g., gel bead) comprises the plurality ofsingle-stranded nucleic acid barcode molecules of FIG. 35C attachedthereto and a plurality of magnetic particles embedded within the solidor semi-solid particle (e.g., gel bead) (see, e.g., FIG. 34 ), whereinthe plurality of magnetic particles comprises, attached thereto, theplurality of nucleic acid barcode molecules of FIG. 35B. In otherembodiments, the solid or semi-solid particle (e.g., gel bead) comprisesthe plurality of nucleic acid barcode molecules of FIG. 35B attachedthereto and a plurality of magnetic particles embedded within the solidor semi-solid particle (e.g., gel bead), wherein the plurality ofmagnetic particles comprises, attached thereto, the plurality of nucleicacid barcode molecules of FIG. 35C. In some embodiments, the barcodesequence in FIG. 35C and the barcode sequence FIG. 35B is the same. Insome embodiments, the barcode sequence in FIG. 35C and the barcodesequence FIG. 35B comprise one or more barcode segments that areidentical. In some embodiments, the barcode sequence in FIG. 35C and thebarcode sequence FIG. 35B are different.

Continuing these embodiments, after partitioning, the single cells (ornuclei) are lysed to release template genomic DNA and template RNA(e.g., cytoplasmic mRNA or nuclear mRNA) in a manner that substantiallymaintains native chromatin organization of the genomic DNA. In certainembodiments, the plurality of nucleic acid barcode molecules of FIG. 35Cand FIG. 35D are releasably attached to the solid or semi-solid particle(e.g., bead, such as a gel bead) and partitions (e.g., droplets orwells) are subjected to conditions to cause release of barcodeoligonucleotide molecules from the solid or semi-solid particle (e.g.,bead) (e.g., depolymerization or degradation of a bead, for example,using a reducing agent such as DTT). Partitions (e.g., droplets orwells) are then subjected to conditions to generate atransposase-nucleic acid complex as described in the aforementionedexamples. Alternatively, in some embodiments, a plurality of pre-formedtransposase-nucleic acid complexes (e.g., FIG. 29A) are partitioned intothe plurality of partitions (e.g., droplets or wells). Partitions arethen subjected to conditions such that the transposase-nucleic acidcomplexes generate double-stranded template genomic DNA fragments.Alternatively, in some embodiments, the transposition reaction isperformed in bulk in intact nuclei which are partitioned such that atleast some partitions (e.g., droplets or wells) comprise (1) a singlecell (or a single nucleus) comprising template genomic DNA fragments andtemplate RNA molecules; (2) a solid or semi-solid particle (e.g., bead,such as a gel bead) comprising (i) a plurality of single-strandednucleic acid barcode molecules as shown in FIG. 35C, and (ii) aplurality of nucleic acid barcode molecules as shown in FIG. 35D; and(3) a plurality of reverse transcriptase molecules. In some embodiments,the nucleic acid barcode molecules of FIG. 35C or FIG. 35D are attachedto a magnetic particle embedded within a solid or semi-solid particle(e.g., bead, such as a gel bead), as previously described. Afterpartitioning, the single nuclei are lysed to release template genomicDNA fragments and template RNA (e.g., cytoplasmic mRNA or nuclear mRNA)molecules. Continuing this embodiment, after fragmentation, templategenomic DNA fragments are processed generally as outlined in FIG. 31 (orFIG. 30B for bulk tagmentation) by performing a linear amplification oftemplate nucleic acid fragments (which in some embodiments, aregap-filled prior to amplification) using nucleic acid barcode moleculesof FIG. 35C (see, e.g., Examples 11 and 12). Using the reversetranscriptase, template RNA molecules comprising a poly-A tail (e.g.,mRNA molecules) are processed with the plurality of nucleic acid barcodemolecules of FIG. 35B to generate barcoded cDNA molecules as generallydescribed elsewhere herein.

Exemplary Scheme 4—scATAC-seq−H 5′ scIZNA-seq Using Linear Amplification

In another barcoding scheme, cells from a cell population of interest(or intact nuclei from cells in a cell population of interest) arepartitioned into a plurality of partitions (e.g., droplets or wells)such that at least some partitions comprise: (1) a single cell (or asingle nucleus) comprising template genomic DNA molecules and templateRNA molecules (e.g., mRNA or nuclear pre-mRNA); (2) a solid orsemi-solid particle (e.g., bead, such as a gel bead) comprising (i) aplurality of single-stranded nucleic acid barcode molecules as shown inFIG. 35C, and (ii) a plurality of nucleic acid barcode molecules asshown in FIG. 35D; (3) a plurality of transposase molecules; (4) aplurality of nucleic acid molecules comprising a transposase endsequence (e.g., FIG. 29A); (5) a plurality of nucleic acid moleculescomprising a poly-T sequence; and (6) a plurality of reversetranscriptase molecules. In some embodiments, the solid or semi-solidparticle is a bead. In some embodiments, the bead is a gel bead. In someembodiments, the solid or semi-solid particle (e.g., gel bead) comprisesthe plurality of single-stranded nucleic acid barcode molecules of FIG.35C attached thereto and a plurality of magnetic particles embeddedwithin the solid or semi-solid particle (e.g., gel bead) (see, e.g.,FIG. 34 ), wherein the plurality of magnetic particles comprises,attached thereto, the plurality of nucleic acid barcode molecules ofFIG. 35D. In other embodiments, the solid or semi-solid particle (e.g.,gel bead) comprises the plurality of nucleic acid barcode molecules ofFIG. 35D attached thereto and a plurality of magnetic particles embeddedwithin the solid or semi-solid particle (e.g., gel bead), wherein theplurality of magnetic particles comprises, attached thereto, theplurality of nucleic acid barcode molecules of FIG. 35C. In someembodiments, the barcode sequence in FIG. 35C and the barcode sequenceFIG. 35D is the same. In some embodiments, the barcode sequence in FIG.35C and the barcode sequence FIG. 35D comprise one or more barcodesegments that are identical. In some embodiments, the barcode sequencein FIG. 35C and the barcode sequence FIG. 35D are different.

Continuing these embodiments, after partitioning, the single cells (ornuclei) are lysed to release template genomic DNA and template RNA(e.g., cytoplasmic mRNA or nuclear mRNA) in a manner that substantiallymaintains native chromatin organization of the genomic DNA. In certainembodiments, the plurality of nucleic acid barcode molecules of FIG. 35Cand FIG. 35D are releasably attached to the solid or semi-solid particle(e.g., bead, such as a gel bead) and partitions (e.g., droplets orwells) are subjected to conditions to cause release of barcodeoligonucleotide molecules from the solid or semi-solid particle (e.g.,bead) (e.g., depolymerization or degradation of a bead, for example,using a reducing agent such as DTT). Partitions (e.g., droplets orwells) are then subjected to conditions to generate atransposase-nucleic acid complex as described in the aforementionedexamples. Alternatively, in some embodiments, a plurality of pre-formedtransposase-nucleic acid complexes (e.g., FIG. 29A) are partitioned intothe plurality of partitions (e.g., droplets or wells). Partitions arethen subjected to conditions such that the transposase-nucleic acidcomplexes generate double-stranded template genomic DNA fragments.Alternatively, in some embodiments, the transposition reaction isperformed in bulk in intact nuclei which are partitioned such that atleast some partitions comprise (1) a single cell (or a single nucleus)comprising template genomic DNA fragments and template RNA molecules;(2) a solid or semi-solid particle (e.g., bead, such as a gel bead)comprising (i) a plurality of single-stranded nucleic acid barcodemolecules as shown in FIG. 35C, and (ii) a plurality of nucleic acidbarcode molecules as shown in FIG. 35D; (3) a plurality of nucleic acidmolecules comprising a poly-T sequence; and (4) a plurality of reversetranscriptase molecules. After partitioning, the single nuclei are lysedto release template genomic DNA fragments and template RNA (e.g.,cytoplasmic mRNA or nuclear mRNA) molecules. Continuing this embodiment,after fragmentation, template genomic DNA fragments are processedgenerally as outlined in FIG. 31 (or FIG. 30B for bulk tagmentation) byperforming a linear amplification of template nucleic acid fragments(which in some embodiments, are gap-filled prior to amplification) usingnucleic acid barcode molecules of FIG. 35C (see, e.g., Examples 11 and12). Template RNA molecules (e.g., mRNA molecules) are processed togenerate 5′ barcoded cDNA molecules as previously described. Forexample, a template RNA molecule comprising a poly-A tail (e.g., mRNA)and a nucleic acid molecule comprising a poly-T sequence are processedusing a reverse transcriptase (e.g., a reverse transcriptase withterminal transferase activity) to generate a cDNA molecule comprisingadditional nucleotides on the 5′ end (e.g., poly-C). The additionalnucleotides on the cDNA hybridize to the additional nucleotides (e.g.,poly-G) on the switch oligo, whereby the switch oligonucleotide sequencein the plurality of nucleic acid barcode molecules of FIG. 35D are usedto further extend the cDNA to generate the 5′ barcoded cDNA molecules.

In the aforementioned embodiments, after barcoding, the barcoded,adapter-flanked DNA fragments and the barcoded cDNA molecules are thenreleased from the partitions (e.g., droplets or wells) to provide areleased mixture and processed in bulk to complete library preparationfor next generation high throughput sequencing (e.g., subjecting thefragments, or derivatives thereof, to one or more reactions (e.g.,nucleic acid amplification) to add functional sequences to facilitateIllumina sequencing). In some embodiments, a first portion of thereleased mixture comprising the adapter-flanked DNA fragments and thebarcoded cDNA is taken and processed in bulk to complete librarypreparation for the barcoded, adapter-flanked DNA fragments while asecond portion of the released mixture is taken and processed in bulk tocomplete library preparation for the barcoded cDNA molecules. In otherembodiments, a first portion of the partitions (e.g., droplets or wells)comprising the barcoded, adapter-flanked DNA fragments and the barcodedcDNA molecules is taken and processed in bulk to complete librarypreparation for the barcoded, adapter-flanked DNA fragments while asecond portion of the partitions (e.g., droplets or wells) comprisingthe barcoded, adapter-flanked DNA fragments and the barcoded cDNAmolecules is taken and processed in bulk to complete library preparationfor the barcoded cDNA molecules. In embodiments that utilize a magneticparticle (e.g., bead), the barcoded template molecules (e.g., barcodedtemplate DNA fragments or barcoded cDNA molecules, or derivativesthereof) attached thereto can be magnetically separated and furtherprocessed to complete library preparation. The fully constructed libraryor libraries are then sequenced according to a suitable next-generationsequencing protocol (e.g., Illumina sequencing).

Example 18 Effect of R1 Sequence Modifications on Barcode Exchange

Nuclei from mice and humans were tagmented and barcoded by ligation(see, e.g., Example 9 and FIGS. 29A-29C) with one of three types ofpartially double-stranded barcodes, mixed, and subsequently subjected todetection in order to assess the effect of each type of barcode on theability to distinguish the different types of nuclei. The three types ofbarcodes included a barcode with a standard R1 sequence (FIG. 37 ; top),a barcode with a shortened R1 sequence compared to the standard R1sequence (FIG. 37 ; middle) and a barcode comprising a uracil in the R1sequence (FIG. 37 ; bottom).

Compared to the separation of the different cell types seen in thebarcodes with standard R1 sequences, the barcodes with the shortened R1sequence and barcodes with a uracil showed greater separation of miceand human nuclei, indicating a reduced rate of barcode exchange (FIGS.38A-38C).

Example 19 Reduction of Mitochondrial DNA Reads Using a Blocking Agent

A plurality of cells is provided in which each cell comprises a templatenucleic acid (e.g., nuclear DNA, such as chromatin) and a non-templatenucleic acid (e.g., mitochondrial DNA). The plurality of cells is lysedin the presence of a cell lysis agent and a blocking agent (e.g., bovineserum albumin) to generate a plurality of lysed cells. A plurality ofnucleic is then separated from the plurality of lysed cells to generatea plurality of biological particles (e.g., nuclei), where a givenbiological particle of the plurality of biological particles comprisesnon-template nucleic acid molecules (e.g., mitochondrial DNA molecules)and template nucleic acid molecules. The template nucleic acid moleculesmay comprise, for example, DNA molecules and RNA molecules. A pluralityof template nucleic acid fragments may then be generated in biologicalparticles of the plurality of biological particles with the aid of atransposase-nucleic acid complex comprising a transposase nucleic acidmolecule and a transposon end nucleic acid molecule. A plurality ofpartitions may then be generated, where a given partition comprises (i)a single biological particle comprising the plurality of templatenucleic acid fragments and template RNA molecules and (ii) a pluralityof first barcode oligonucleotide molecules (e.g., first nucleic acidbarcode molecules) comprising a barcode sequence. The partitions mayfurther comprise (iii) a plurality of second barcode oligonucleotidemolecules (e.g., second nucleic acid molecules) comprising a barcodesequence and a capture sequence; and (iv) a plurality of reversetranscriptase molecules. The partitions may be, for example, droplets orwells.

Barcoded template DNA fragments may then be generated within thepartitions using barcode oligonucleotide molecules of the plurality offirst barcode oligonucleotide molecules and template nucleic acidfragments of the plurality of template DNA fragments. If template RNAmolecules are present, barcoded cDNA molecules may be generated from thetemplate RNA molecules by reverse transcription using barcodeoligonucleotide molecules of the plurality of second barcodeoligonucleotide molecules. In some cases, the biological particles maybe nuclei, which may be isolated from lysed cells by, e.g., washing thelysed cells. In some cases, the method further comprises generatingsequencing reads. Application of the method may reduce the fraction ofsequencing reads deriving from non-template nucleic acid molecules(e.g., mitochondrial DNA molecules) relative to the total number ofsequencing reads generated.

Example 20 Reduction of Mitochondrial DNA Reads in Partitions

A plurality of partitions (e.g., droplets or wells) is provided. A givenpartition of the plurality of partitions comprises: (i) a singlebiological particle (e.g., cell) from a plurality of biologicalparticles (e.g., cells), wherein the single biological particlecomprises template nucleic acid molecules (e.g., nuclear DNA molecules,such as chromatin) and non-template DNA molecules (e.g., mitochondrialDNA molecules); (ii) a plurality of barcode oligonucleotide molecules(e.g., nucleic acid barcode molecules) comprising a barcode sequence;(iii) a plurality of transposon end oligonucleotide molecules comprisinga transposon end sequence; and (iv) a plurality of transposasemolecules. The given partition may further comprise reversetranscriptases and a plurality of second barcode oligonucleotidemolecules (e.g., second nucleic acid barcode molecules) comprising abarcode sequence and a capture sequence. Barcoded template nucleic acidfragments and barcoded non-template nucleic acid fragments may begenerated as described in the preceding examples. One or morenon-template nucleic acid fragments of the plurality of non-template DNAfragments, or derivatives thereof, may then be cleaved using (i) one ormore guide ribonucleic acid molecules (gRNAs) targeted to the one ormore non-template nucleic acid fragments, and (ii) a clustered regularlyinterspaced short palindromic (CRISPR) associated (Cas) nuclease. Insome cases, the method may reduce the amount of fragments and/orbarcoded fragments comprising and/or deriving from non-template nucleicacid molecules. For example, the method may reduce the total number ofnon-template nucleic acid fragments and barcoded non-template nucleicacid fragments, e.g., in a mixture comprising one or more of templatenucleic acid fragments, barcoded template nucleic acid fragments,non-template nucleic acid fragments, and barcoded non-template nucleicacid fragments. In some cases, the endonuclease may be Cas9, such asrecombinant Cas9. In some cases, the non-template nucleic acid fragmentmay comprise a mitochondrial DNA or RNA fragment. In some cases, thetemplate nucleic acid fragment may comprise a nuclear DNA fragment.

Example 21 Comparison of Linear Amplification and Ligation Methods

Sample libraries may be generated using any of the methods described inthe preceding examples. In some cases, a library may be generated usinga linear amplification method. In other cases, a library may begenerated using a ligation method. A linear amplification method maycomprise, within a partition, generating template nucleic acid fragmentsand performing a gap filling extension process within the partition.Linear amplification may then be performed using, for example, a heatresistant polymerase. Barcodes may be incorporated into sequencescomprising template nucleic acid sequences, or complements thereof. Thebarcoded nucleic acid fragments may then be released from theirrespective partitions into the bulk for additional processing including,for example, PCR. A ligation method may comprise, within a partition,generating template nucleic acid fragments and ligating barcodes to thefragments with a ligase such as a T4 DNA ligase, thereby generatingbarcoded nucleic acid fragments. The barcoded nucleic acid fragments maythen be released from their respective partitions into the bulk foradditional processing including, for example, gap filling and additionof additional sequences.

In order to compare linear amplification and ligation methods, variousmouse and human cells were processed according to the methods describedherein.

FIG. 40 shows a table comparing sequencing metrics generated fromdifferent replicate sample libraries (“A” and “B”) generated usingeither linear amplification or ligation. “A” replicates representreactions done in the same conditions, but using a different user, while“B” replicates represent reactions done in the same conditions by thesame user.

FIGS. 41A-41B illustrate exemplary amounts of detected mouse or humancells and the inferred doublet rate observed from the analysis ofsequencing reads (FIG. 40 ) from a linear amplification ATAC-seq library(using 75 nM, 150 nM, or 250 nM of barcode primer) or ligation-basedATAC-seq library (using the barcode molecules of FIG. 37 ). FIG. 41Aillustrates differences in the number of mouse or human cells detectedusing either a linear amplification or ligation-based ATAC-seq method.FIG. 41B illustrate differences in inferred doublet rate detected usingeither a linear amplification or ligation-based ATAC-seq method. Asshown in FIG. 41B, linear amplification demonstrated a higher doubletrate than ligation.

FIG. 42 illustrates a comparison of the sensitivity of sequencing readsobtained from various ATAC-seq libraries as measured by the medianfragments per cell barcode. The sensitivity of the linear amplificationand ligation methods of library preparation (36k reads/cell) is comparedto the methods described in Buenrostro, et al., Nature, 2015 Jul. 23;523(7561):48690 using a programmable microfluidics platform (Fluidigm).As shown in FIG. 42 , ligation provides higher sensitivity than linearamplification.

As shown in FIG. 43 , a library prepared by ligation sequenced toincreased depth (30M reads to 800M reads) demonstrates a significantincrease in the sensitivity (as measured by median fragments per cellbarcode) and reduced noise (as measured by fraction of non-duplicatewasted reads) relative to the lower depth library.

FIG. 48 shows a comparison of exemplary sequencing metrics obtained fromlinear amplification-based ATAC-seq libraries prepared using differentpolymerases: a Phusion® DNA polymerase, a KAPA HiFi DNA polymerase (incombination with betaine), a Deep Vent® DNA polymerase, as well as alibrary prepared by ligation.

“Wasted” Reads

As shown in FIG. 44 , non-duplicate “wasted” reads may be generatedwhile performing the methods described herein. FIG. 44 illustrates anexemplary comparison of the total noise (non-duplicate wasted reads(“Non-dups”) and mitochondrial-based reads (“Mito”)) in librariesprepared using linear amplification or ligation-based ATAC-seq methods.FIGS. 45A-45B provide an exemplary illustration of the breakdownsequencing reads generated by different library preparation methods.FIG. 45A shows an illustration of the breakdown of reads generated by alinear amplification ATAC-seq library. FIG. 45B shows an illustration ofthe breakdown of reads generated by a ligation-based ATAC-seq library.As described above, reads attributable to mitochondrial nucleic acidsmay be removed by the use of blocking agents such as bovine serumalbumin and/or through the use of Cas9 and targeted gRNAs.

Insert Size Distribution

Linear amplification and ligation library preparation methods were alsocompared to examine insert size distributions. FIGS. 46A-46B illustratean exemplary comparison of read pairs showing the periodicity ofnucleosomes generated from ATAC-seq libraries prepared using either athese schemes. Nucleosome free fragments are typically observed below200 bp in length, fragments indicative of a nucleosome periodicity ofone are approximately 200 bp in length, fragments indicative of anucleosome periodicity of two are approximately 400 bp in length,fragments indicative of a nucleosome periodicity of two are 600 bp inlength, and so forth. Bulk enrichment

As shown in FIGS. 47A-47B, linear amplification shows a higherenrichment of transcription start sites (TSS) than ligation due to abias for shorter insert fragments. A difference may also be observedbetween linear amplification and ligation methods for CTCF(CCCTC-binding factor) sites.

Example 22 Analysis of Nucleic Acids in Peripheral Blood MononuclearCell Samples

Peripheral blood mononuclear cells (PBMCs) are subjected to single cellRNA-seq and ATAC-seq analyses as described herein.

FIG. 51A shows an exemplary scatterplot produced using t-DistributedStochastic Neighbor Embedding (tSNE), allowing visualization of RNAtranscripts of different subpopulations cell types in a peripheral bloodmononuclear cell (PBMC) sample. FIG. 51B shows an exemplary scatterplotproduced using t-Distributed Stochastic Neighbor Embedding (tSNE),allowing visualization of ATAC-seq data of different subpopulations celltypes in a peripheral blood mononuclear cell (PBMC) sample.

Example 23 Comparison of ATAC-seq Methods

FIG. 50 illustrates protocols for ATAC-seq analyses as described hereincompared to data from (1) typical high quality traditional bulk ATAC-seqprotocols; (2) Cusanovich, et al., Science, 2015 May 22;348(6237):910-14; (3) Buenrostro, et al., Nature, 2015 Jul. 23;523(7561):486-90; (4) ideal sequencing metrics from an ATAC-seqexperiment; and (5) data obtained using the methods described herein(“10×”).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method of processing a sample, comprising: (a)contacting a plurality of cells or cell nuclei comprising chromatin witha plurality of transposase nucleic acid complexes to generate a cell orcell nucleus comprising a tagged fragment of genomic deoxyribonucleicacid (DNA); (b) partitioning said plurality of cells or cell nuclei anda plurality of barcode sequences into a plurality of partitions, whereina partition of said plurality of partitions comprises: (i) said cell orcell nucleus comprising said tagged fragment of genomic DNA; (ii) afirst barcode oligonucleotide molecule comprising a first barcodesequence; (iii) a second barcode oligonucleotide molecule comprising asecond barcode sequence; and (iv) a reverse transcriptase; (c) usingsaid first barcode oligonucleotide molecule and said tagged fragment ofgenomic DNA to generate a first barcoded molecule comprising (1) asequence of said tagged fragment of genomic DNA, and (2) said firstbarcode sequence, or a reverse complement thereof; and (d) using saidsecond barcode oligonucleotide molecule, said reverse transcriptase, anda ribonucleic acid (RNA) molecule of said cell or cell nucleus togenerate a second barcoded molecule comprising (1) a complementary DNA(cDNA) sequence of said RNA molecule and (2) said second barcodesequence, or a reverse complement thereof.
 2. The method of claim 1,wherein said second barcode oligonucleotide molecule comprises atemplate switching sequence and wherein (d) comprises performing atemplate switching reaction to generate said second barcoded molecule.3. The method of claim 2, wherein said partition further comprises aprimer molecule comprising a sequence complementary to said RNAmolecule, wherein (d) comprises hybridizing said primer molecule to saidRNA molecule, reverse transcribing said RNA molecule to generate a cDNAmolecule comprising said cDNA sequence, and using said templateswitching sequence to perform said template switching reaction, andwherein said template switching reaction comprises using said secondbarcode oligonucleotide molecule as a template to extend said cDNAmolecule, thereby generating said second barcoded molecule.
 4. Themethod of claim 3, wherein said sequence complementary to said RNAmolecule is a poly-T sequence.
 5. The method of claim 3, wherein saidsequence complementary to said RNA molecule is a random sequence.
 6. Themethod of claim 3, wherein said template switching sequence comprises afirst poly-nucleotide sequence, wherein said reverse transcriptasecomprises terminal transferase activity and adds a secondpoly-nucleotide sequence to said cDNA molecule, wherein said firstpoly-nucleotide sequence is complementary to said second poly-nucleotidesequence, and wherein said first poly-nucleotide sequence hybridizes tosaid second poly-nucleotide sequence, thereby facilitating said templateswitching reaction.
 7. The method of claim 6, wherein said firstpoly-nucleotide sequence comprises a poly-G sequence and wherein saidsecond poly-nucleotide sequence comprises a poly-C sequence.
 8. Themethod of claim 6, wherein said first poly-nucleotide sequence is aribonucleic acid sequence.
 9. The method of claim 1, wherein said secondbarcode oligonucleotide molecule comprises a poly-T sequence, whereinsaid RNA molecule is an mRNA molecule, and wherein (d) compriseshybridizing said poly-T sequence to said mRNA molecule and performing areverse transcription reaction to generate said second barcodedmolecule.
 10. The method of claim 2, wherein (c) comprises ligating saidfirst barcode oligonucleotide molecule to said tagged fragment ofgenomic DNA to generate said first barcoded molecule.
 11. The method ofclaim 9, wherein (c) comprises ligating said first barcodeoligonucleotide molecule to said tagged fragment of genomic DNA togenerate said first barcoded molecule.
 12. The method of claim 2,wherein (c) comprises hybridizing said first barcode oligonucleotidemolecule to said tagged fragment of genomic DNA and performing anextension reaction to generate said first barcoded molecule.
 13. Themethod of claim 9, wherein (c) comprises hybridizing said first barcodeoligonucleotide molecule to said tagged fragment of genomic DNA andperforming an extension reaction to generate said first barcodedmolecule.
 14. A method of processing a sample, comprising: (a)contacting a plurality of cells or cell nuclei comprising chromatin witha plurality of transposase nucleic acid complexes to generate a cell orcell nucleus comprising a tagged fragment of genomic deoxyribonucleicacid (DNA); (b) partitioning said plurality of cells or cell nuclei anda plurality of beads into a plurality of partitions, wherein a partitionof said plurality of partitions comprises: (i) said cell or cell nucleuscomprising said tagged fragment of genomic DNA; (ii) a bead of saidplurality of beads, wherein said bead comprises (1) a first barcodeoligonucleotide molecule comprising a first barcode sequence; and (2) asecond barcode oligonucleotide molecule comprising a template switchingsequence; (iii) a primer molecule; and (iv) a reverse transcriptase; (c)using said first barcode oligonucleotide molecule and said taggedfragment of genomic DNA to generate a first barcoded molecule comprising(1) a sequence of said tagged fragment of genomic DNA, and (2) saidfirst barcode sequence, or a reverse complement thereof; and (d)hybridizing said primer molecule to a ribonucleic acid (RNA) molecule ofsaid cell or cell nucleus, reverse transcribing said RNA molecule togenerate a cDNA molecule, and using said template switching sequence toperform a template switching reaction, wherein said template switchingreaction comprises using said second barcode oligonucleotide molecule asa template to extend said cDNA molecule, thereby generating a secondbarcoded molecule comprising (1) a complementary DNA (cDNA) sequence ofsaid RNA molecule and (2) a reverse complement of said second barcodesequence.
 15. The method of claim 14, further comprising processing saidfirst barcoded molecule and said second barcoded molecule to generate asequencing library.
 16. The method of claim 15, further comprisingsequencing said sequencing library to obtain sequence information ofsaid first barcoded molecule and said second barcoded molecule.
 17. Themethod of claim 1, wherein said first barcode sequence and said secondbarcode sequence are the same; or wherein said first barcode sequenceand said second barcode sequence are different and an associationbetween said first barcode sequence and said second barcode sequence isknown.
 18. The method of claim 14, wherein said first barcode sequenceand said second barcode sequence are the same; or wherein said firstbarcode sequence and said second barcode sequence are different and anassociation between said first barcode sequence and said second barcodesequence is known.
 19. The method of claim 14, wherein said bead is agel bead.
 20. The method of claim 19, wherein said gel bead is adegradable gel bead.
 21. The method of claim 14, wherein said firstbarcode oligonucleotide molecule and said second barcode oligonucleotidemolecule are attached to said bead via a labile bond.
 22. The method ofclaim 21, further comprising, subsequent to (b), applying a stimulus tocleave said labile bond.